B12 dependent dehydratases with improved reaction kinetics

ABSTRACT

Sequences of B 12 -dependent dehydratases with improved reaction kinetics are presented. Use of these B 12 -dependent dehydratases reduce the rate of the enzyme&#39;s suicide inactivation in the presence of glycerol and 1,3-propanediol. The enzymes were created using error-prone PCR and oligonucleotide-directed mutagenesis to target the DhaB1 gene, which encodes the α-subunit of glycerol dehydratase. Mutants with improved reaction kinetics were rapidly identified using high throughput assays.

FIELD OF INVENTION

The present invention relates to the field of molecular biology and theuse of B₁₂-dependent dehydratases to produce 1,3-propanediol. Morespecifically, it describes methods to create and select B₁₂-dependentdehydratases with improved reaction kinetics, such that the rate ofenzyme inactivation is reduced.

BACKGROUND

1,3-Propanediol has utility in a number of applications, including as astarting material for producing polyesters, polyethers, andpolyurethanes. Methods for producing 1,3-propanediol include bothtraditional chemical routes and biological routes. Biological methodsfor producing 1,3-propanediol have been recently described (Zeng et al.,Adv. Biochem. Eng. Biotechnol., 74:239-259 (2002)). Biologicallyproducing 1,3-propanediol requires glycerol as a substrate for atwo-step sequential reaction. First, a dehydratase (typically a coenzymeB₁₂-dependent dehydratase) converts glycerol to an intermediate,3-hydroxypropionaldehyde (3-HP). Then, 3-HP is reduced to1,3-propanediol by an NADH— (or NADPH) dependent oxidoreductase (SeeEquations 1 and 2).Glycerol→3-HP+H₂O  (Equation 1)3-HP+NADH+H⁺→1,3-Propanediol+NAD⁺  (Equation 2)The 1,3-propanediol is not metabolized further and, as a result,accumulates in high concentration in the media.

Typically, glycerol is used as the starting material for biologicallyproducing 1,3-propanediol. However, glucose and other carbohydrates alsoare suitable substrates for 1,3-propanediol production. Specifically,Laffend et al. (WO 96/35796; U.S. Pat. No. 5,686,276) disclose a methodfor producing 1,3-propanediol from a carbon substrate other thanglycerol or dihydroxyacetone (especially, e.g., from glucose), using asingle microorganism comprising a dehydratase activity. Emptage et al.,(WO 01/012833) describe a significant increase in titer (grams productper liter) obtained by virtue of a non-specific catalytic activity(distinguished from the 1,3-propanediol oxidoreductase encoded by dhaT)to convert 3-HP to 1,3-propanediol. Payne et al. (U.S. 60/374,931)disclose specific vectors and plasmids useful for biologically producing1,3-propanediol. Cervin et al. (U.S. 60/416,192) disclose improved E.coli strains for high yield production of 1,3-propanediol. WO 96/35796,WO 01/012833, U.S. 60/374,931, and U.S. 60/416,192 are incorporated byreference in the instant specification as though set forth in theirentirety herein.

The enzymes responsible for converting glycerol to 3-HP are largelycoenzyme B₁₂-dependent enzymes, known as coenzyme B₁₂-dependent glyceroldehydratases (E.C. 4.2.1.30) and coenzyme B₁₂-dependent dioldehydratases (E.C. 4.2.1.28). These distinct, but related, coenzymeB₁₂-dependent enzymes are well studied in terms of their molecular andbiochemical properties. Genes for coenzyme B₁₂-dependent dehydrataseshave been identified, for example, in Klebsiella pneumoniae, Citrobacterfreundii, Clostridium pasteurianum, Salmonella typhimurium, Klebsiellaoxytoca, and Lactobacillus collinoides (Toraya, T., In MetalloenzymesInvolving Amino Acid-Residue and Related Radicals; Sigel, H. and Sigel,A., Eds.; Metal Ions in Biological Systems; Marcel Dekker: New York,1994; Vol. 30, pp 217-254; Daniel et al., FEMS Microbiology Reviews22:553-566 (1999); and Sauvageot et al., FEMS Microbiology Letters 209:69-74 (2002)).

Although there is wide variation in the gene nomenclature used in theliterature, in each case the coenzyme B₁₂-dependent dehydratase iscomposed of three subunits: the large or “α” subunit, the medium or “β”subunit, and the small or “γ” subunit. These subunits assemble in anα₂β₂γ₂ structure to form the apoenzyme. Coenzyme B₁₂ (the activecofactor species) binds to the apoenzyme to form the catalyticallyactive holoenzyme. Coenzyme B₁₂ is required for catalytic activity as itis involved in the radical mechanism by which catalysis occurs.

Biochemically, both coenzyme B₁₂-dependent glycerol and coenzymeB₁₂-dependent diol dehydratases are known to be subject tomechanism-based suicide inactivation by glycerol and other substrates(Daniel et al., supra; Seifert, et al., Eur. J. Biochem. 268:2369-2378(2001)). In addition, inactivation occurs via interaction of theholoenzyme with high concentrations of 1,3-propanediol. Inactivationinvolves cleavage of the cobalt-carbon (Co—C) bond of the coenzyme B₁₂cofactor, leading to the formation of 5′-deoxyadenosine and an inactivecobalamin species. The inactive cobalamin species remains tightly boundto the dehydratase; dissociation does not occur without the interventionof coenzyme B₁₂-dependent dehydratase reactivation factors (“dehydratasereactivation factors”). This inactivation can significantly decrease thereaction kinetics associated with 3-HP formation and, thus, indirectlydecrease 1,3-propanediol production.

The effects of coenzyme B₁₂-dependent dehydratase inactivation can bepartially overcome. For example, inactivation can be overcome by relyingon those proteins responsible for reactivating the dehydratase activity.Dehydratase reactivation factors have been described in: WO 98/21341(U.S. Pat. No. 6,013,494); Daniel et al. (supra); Toraya and Mori (J.Biol. Chem. 274:3372 (1999)); and Tobimatsu et al. (J. Bacteriol.181:4110 (1999)). Reactivation occurs in a multi-step process.Initially, interaction of inactivated coenzyme B₁₂-dependent dehydratasewith dehydratase reactivation factors, in an ATP-dependent process,results in the release of the tightly bound inactive cobalamin speciesto produce apoenzyzme. Subsequently, the dehydratase apoenzyme may bindcoenzyme B₁₂ to re-form the catalytically active holoenzyme and theinactive cobalamin species may be regenerated (by enzymatic action, in aseparate ATP-dependent process) to coenzyme B₁₂. Depending solely ondehydratase reactivation factors to restore dehydratase activity isinherently limited, however, since both the dehydratase reactivation andthe coenzyme B₁₂ regeneration processes require ATP. These ATP-dependentprocesses represent a significant energetic burden to the process ofconverting glycerol to 3-HP, particularly if a subsequent reaction of3-HP to 1,3-propanediol is present and the 1,3-propanediol concentrationis high.

Alternatively, it is possible to either increase the amount of coenzymeB₁₂ added to a medium during 1,3-propanediol production or to supplementthe culture media with vitamin B₁₂ (which is converted to coenzyme B₁₂in vivo) to supply additional coenzyme B₁₂ to microorganisms. However,in both cases, the cost of these additions may significantly interferewith process economics.

Croux et al. (WO 01/04324 A1) have addressed the problems associatedwith coenzyme B₁₂-dependent dehydratases by developing a process forproducing 1,3-propanediol using a recombinant microorganism thatexpresses a coenzyme B₁₂-independent dehydratase. However, theusefulness of this solution may be limited by the ability ofB₁₂-independent dehydratases to function under certain preferred processconditions (e.g. under aerobic conditions (Hartmanis and Stadman, Arch.Biochem. Biophys. 245:144-52 (1986)).

In principle, it should be possible to isolate coenzyme B₁₂-dependentdehydratases with reduced inactivation kinetics from naturally occurringmicrobial strains. Reduced inactivation kinetics would increase theturnover ratio (mol product/mol holoenzyme) of coenzyme B₁₂-dependentdehydratase in a microbial host, and thus, reduce the dehydratase andcoenzyme B₁₂ demand. This approach would also reduce the energy neededto maintain that level of dehydratase and coenzyme B₁₂. However, inpractice, the diversity of coenzyme B₁₂-dependent dehydratases has beenfound to be limited with respect to inactivation kinetics.

The problem to be solved, therefore, is that currently availablecoenzyme B₁₂-dependent dehydratase enzymes are unable to provide thereaction kinetics needed for industrial applications for the productionof industrial compounds.

SUMMARY OF THE INVENTION

Applicants have provided a method of screening for B₁₂-dependentdehydratases having improved reaction kinetics, comprising:

-   -   (a) contacting a B₁₂-dependent dehydratase holoenzyme with a        mixture comprising glycerol and 1,3-propandiol, wherein the        1,3-propanediol is at least 25 mM;    -   (b) screening the B₁₂-dependent dehydratase holoenzyme to        estimate the turnover ratio of the B₁₂-dependent dehydratase.

The screening step of the method further comprises the steps of:

-   -   a) growing a cell on a fermentable carbon substrate that does        not include glycerol, wherein the cell does not have a source of        coenzyme B₁₂, dehydratase reactivation factor, or endogenous        B₁₂-dependent dehydratase;    -   b) permeabilizing the cell;    -   c) adding a mixture of coenzyme B₁₂, glycerol, and at least 25        mM 1,3-propanediol to the permeabilized cell of step (b);    -   d) quantitating 3-hydroxypropionaldehyde produced in step (c),        wherein the quantitating is a measurement selected from the        group consisting of T1, T2, and T(600).

The invention further includes a nucleic acid sequence encoding aB₁₂-dependent mutant dehydratase selected from the group consisting ofSEQ ID Nos:40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96,100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 143, 147, 151,155, 159, 163, 167, 171, 175, 179, 182, 186, 189, 192, 195, 198, 201,204, 208, 212, 215, 218, 221, 225, 229, 233, 237, 241, 245, 249, 253,257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 304, 307,310, 313, 316, 319, 322, 325, 328, 331, 334, and 337. More preferredB₁₂-dependent mutant dehydratases encoded by the nucleic acid sequenceare selected from the group consisting of: SEQ ID NO: 40, 44, 48, 52,56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116,120, 124, 128, 132, 136, 140, 143, 147, 151, 155, 159, 163, 167, 171,175, 179, 182, 186, 189, 192, 195, 198, 201, 204, 208, 212, 215, 218,221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273,277, 281, 285, 289, 293, 297, 301, 304, 307, 310, 313, 316, 319, 322,325, 328, 331, 334, and 337. More preferred nucleic acid sequencesencoding a B₁₂-dependent mutant dehydratase comprise an α-β subunitfusion that are selected from the group consisting of SEQ ID NOs:140,179, 186, 189, 192, 195, 198, 201, 212, 215, 218, 301, 304, 307, 310,313, 316, 319, 322, 325, 328, 331, 334, and 337. Most preferred nucleicacid sequences encoding a B₁₂-dependent mutant dehydratase comprise anα-β subunit fusion selected from the group consisting of SEQ ID NOs:313,322, and 328.

-   -   The invention also includes the nucleic acid sequences, further        comprising a linker sequence between the α and β subunits of the        α-β subunit fusion, wherein the linker sequence is selected from        the group consisting of SEQ ID NO:18 and SEQ ID NO:19.

The invention also includes a method for creating B₁₂-dependentdehydratase mutants having improved reaction kinetics, comprising:

-   -   a) contacting a B₁₂-dependent dehydratase holoenzyme comprising        hotspot 1 or hotspot 2 with a mutating agent;    -   b) screening the mutants produced in step A) for improved kcat        and/or stability; and    -   c) repeating steps a) and b).

7. An further method of the invention for identifying B₁₂-dependentdehydratase with improved reaction kinetics relative to a referencedehydratase comprises

-   -   a) contacting a dehydratase holoenzyme with 5-10 mM glycerol        and/or 10-300 mM 1,3-propanediol;    -   b) measuring at least two time points in a high throughput assay        sufficiently separated to estimate k_(cat) and total enzyme        turnover;    -   c) selecting B₁₂-dependent mutants having improved reaction        kinetics relative to the reference dehydratase.

A still further method of the invention for identifying B₁₂-dependentdehydratase with improved reaction kinetics relative to a referencedehydratase comprises

-   -   a) contacting a dehydratase holoenzyme with 5-50 mM glycerol        and >300 mM 1,3-propanediol;    -   b) measuring one time point in a high throughput assay        sufficiently separated from T0 to estimate the total enzyme        turnover number; and    -   c) selecting B₁₂-dependent mutants having improved reaction        kinetics relative to the reference dehydratase.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

FIG. 1 shows the time course of a typical GDH reaction, performed eitherin the presence of glycerol or in the presence of glycerol and1,3-propanediol.

FIG. 2 graphically illustrates results of a typical follow-up assay asdescribed in Example 6.

FIG. 3 shows the distribution of mutants containing a single-pointmutation (relative to wild-type GDH) on the α-subunit.

FIG. 4 shows the distribution of mutants containing multiple-pointmutations (relative to wild-type GDH) on the α-subunit.

FIG. 5 shows the principle of the recombinogenic extension method usingunpaired primers.

Applicants have provided 346 sequences in conformity with Rules for theStandard Representation of Nucleotide and Amino Acid Sequences in patentapplications (Annexes I and II to the Decision of the President of theEPO, published in Supplement No. 2 to OJ EPO, December 1992), with 37C.F.R. 1.821-1.825 and Appendices A and B (Requirements for ApplicationDisclosures Containing Nucleotides and/or Amino Acid Sequences) withWorld Intellectual Property Organization (WIPO) Standard ST.25 (1998)and the sequence listing requirements of the EPO and PCT (Rules 5.2 and49.5 (a-bis), and Section 208 and Annex C of the AdministrativeInstructions). The Sequence Descriptions contain the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IYUB standards describedin Nucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (2):345-373 (1984), which are herein incorporated byreference.

SEQ ID NO:1 is a 12.1 kB EcoRI-Sall fragment containing the wild-typeGDH isolated from Klebsiella pneumoniae ATCC 25955 (Emptage et al., WO01/012833 A2). The wild-type GDH is encoded by the α-subunit (bp7044-8711), the β-subunit (bp 8724-9308), and the γ-subunit (bp9311-9736). The amino acid sequences of the α, β, and γ-subunits of GDHare provided as SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively.

SEQ ID NOs: 5 and 6 encode primers DHA-F1 and DHA-R1, respectively, usedfor cloning GDH from plasmid pGH20.

SEQ ID NO: 7 encodes reverse primer DHA-R2, utilized for cloning theentire α- and a portion of the β-subunit of GDH.

SEQ ID NOs: 8-14 encode primers pGD20RM-F1, pGD20RM-R1, TB4BF, TB4BR,pGD20RM-F2, pGD20RM-R2, and GD-C respectively, used for regional randommutagensis of the α-subunit.

SEQ ID NOs: 15-17 encode degenerate primers pGD20RM-F3, TB4B-R1, andpGD20RM-R4, respectively, used for preparation of the regional randommutant libraries.

SEQ ID NO: 18 encodes the linker between the α- and β-subunits of fusionprotein Sma3002. SEQ ID NO: 19 encodes the linker between the α- andβ-subunits of fusion protein Xba3009.

SEQ ID NOs: 20-25 encode primers 2-F4-F1, 2-F4-R1, 12-B1-F1, 12-B1-R1,16-H5-F1, and 16-H5-R1, respectively, utilized for synthesis of secondgeneration mutant GDHs.

SEQ ID NOs: 26-29 encode primers 1-E1-F1, 1-E1-R1, 22-G7-F1, and22-G7-R1, respectively, used for creating the pure fusion mutants 1-E1and 22-G7.

SEQ ID NOs: 30-39 encode primers 7A-C1-F1, 7A-C1-R1, 7C-A5-F1, 7C-A5-R1,8-C9-F1, 8-C9-R1, 9-D7-F1, 9-D7-R1, 10-G6-F1, and 110-G6-R1,respectively, used for synthesis of Sma3002-derived mutants.

Mutant enzymes derived from the wild-type GDH are assigned the followingSEQ ID NOs, according to their respective nucleic acid sequences andamino acid sequences (Table 1): TABLE 1 Full Length Mutant GDHs andtheir Respective SEQ ID NOs Mutagenesis Method Used Nucleic Acid AminoAcid Strain to Create SEQ ID NO SEQ ID NO Xba3007 1^(st) Round Random 4041, 42, 43 Xba3029 1^(st) Round Random 44 45, 46, 47 Xba3004 1^(st)Round Random 48 49, 50, 51 Xba3025 1^(st) Round Random 52 53, 54, 55Xba3038 1^(st) Round Random 56 57, 58, 59 Xba3030 1^(st) Round Random 6061, 62, 63 Xba3006 1^(st) Round Random 64 65, 66, 67 Xba3031 1^(st)Round Random 68 69, 70, 71 Xba3017 1^(st) Round Random 72 73, 74, 75Xba3005 1^(st) Round Random 76 77, 78, 79 Xba3033 1^(st) Round Random 8081, 82, 83 Xba3032 1^(st) Round Random 84 85, 86, 87 Xba3018 1^(st)Round Random 88 89, 90, 91 Xba3014 1^(st) Round Random 92 93, 94, 95Xba3024 1^(st) Round Random 96 97, 98, 99 Xba3026 1^(st) Round Random100 101, 102, 103 Sma3009 1^(st) Round Random 104 105, 106, 107 Sma30101^(st) Round Random 108 109, 110, 111 Sma3014 1^(st) Round Random 112113, 114, 115 Sma3008 1^(st) Round Random 116 117, 118, 119 Sma30011^(st) Round Random 120 121, 122, 123 PpuMI001 1^(st) Round RegionalRandom 124 125, 126, 127 PpuMI002 1^(st) Round Regional Random 128 129,130, 131 PpuMI005 1^(st) Round Regional Random 132 133, 134, 135RsrII001 1^(st) Round Regional Random 136 137, 138, 139 Sma3002 1^(st)Round Random 140 141, 142 Sma3003 1^(st) Round Random 143 144, 145, 146Xba3015 1^(st) Round Random 147 148, 149, 150 Xba3008 1^(st) RoundRandom 151 152, 153, 154 Xba3016 1^(st) Round Random 155 156, 157, 158Xba3020 1^(st) Round Random 159 160, 161, 162 Xba3037 1^(st) RoundRandom 163 164, 165, 166 Xba3036 1^(st) Round Random 167 168, 169, 1704BR1001 1^(st) Round Regional Random 171 172, 173, 174 Xba3010 1^(st)Round Random 175 176, 177, 178 Xba3009 1^(st) Round Random 179 180, 181Xba3023 1^(st) Round Random 182 183, 184, 185 2-F4 2^(nd) RoundRationale Design 186 187, 188 12-B1 2^(nd) Round Rationale Design 189190, 191 13-B7 2^(nd) Round Rationale Design 192 193, 194 16-H5 2^(nd)Round Rationale Design 195 196, 197 1-E1 2^(nd) Round Rationale Design198 199, 200 22-G7 2^(nd) Round Rationale Design 201 202, 203 7A-C12^(nd) Round Rationale Design 204 205, 206, 207 7C-A5 2^(nd) RoundRationale Design 208 209, 210, 211 8-C9 2^(nd) Round Rationale Design212 213, 214 9-D7 2^(nd) Round Rationale Design 215 216, 217 10-G62^(nd) Round Rationale Design 218 219, 220 21-D10 3^(rd) Round RationaleDesign 221 222, 223, 224 20-B9 3^(rd) Round Rationale Design 225 226,227, 228 18-D7 3^(rd) Round Rationale Design 229 230, 231, 232 17-F63^(rd) Round Rationale Design 233 234, 235, 236 15-E4 3^(rd) RoundRationale Design 237 238, 239, 240 KG002 1^(st) Round Random 241 242,243, 344 KG003 1^(st) Round Random 245 246, 247, 248 KG004 1^(st) RoundRandom 249 250, 251, 252 KG005 1^(st) Round Random 253 254, 255, 256KG006 1^(st) Round Random 257 258, 259, 260 KG007 1^(st) Round Random261 262, 263, 264 KG010 1^(st) Round Random 265 266, 267, 268 KG0111^(st) Round Random 269 270, 271, 272 KG012 1^(st) Round Random 273 274,275, 276 KG014 1^(st) Round Random 277 278, 279, 280 KG016 1^(st) RoundRandom 281 282, 283, 284 KG017 1^(st) Round Random 285 286, 287, 288KG021 1^(st) Round Random 289 290, 291, 292 KG023 1^(st) Round Random293 294, 295, 296 KG001 1^(st) Round Random 297 298, 299, 300 GDH-SM1-Site-saturation 301 302, 303 G11 GDH-SM2- Site-saturation 304 305, 306B11 GDH-SM3- Site-saturation 307 308, 309 D2 GDH-SM4- Site-saturation310 311, 312 H2 SHGDH37 Unpaired Primers 313 314, 315 SHGDH51 UnpairedPrimers 316 317, 318 SHGDH12 Unpaired Primers 319 320, 321 SHGDH22Unpaired Primers 322 323, 324 SHGDH38 Unpaired Primers 325 326, 327SHGDH24 Unpaired Primers 328 329, 330 SHGDH43 Unpaired Primers 331 332,333 SHGDH25 Unpaired Primers 334 335, 336 SHGDH29 Unpaired Primers 337338, 339

SEQ ID NOS:340-342 encode the primers T53-SM, L509-SM, and V224-SM,respectively.

SEQ ID Nos:343 and 344 encode the primers GDHM-F1 and GDHM-R1,respectively.

SEQ ID Nos:345 and 346 encode the primers GDHM-F2 and GDHM-R2,respectively.

DETAILED DESCRIPTION OF THE INVENTION

The Applicants have solved the stated problem by providing engineeredcoenzyme B₁₂-dependent dehydratases with improved reaction kinetics(i.e., providing an increased total turnover number in the presence ofglycerol and 1,3-propanediol) for use in industrial applications,specifically for producing 3-hydroxypropionaldehyde and 1,3-propanediol.Created by techniques of mutagenesis, these dehydratases arecharacterized by either having an increased k_(cat) and/or a decreasedrate of enzyme inactivation, relative to the wild-type enzyme from whichthey were created.

The engineered coenzyme B₁₂-dependent dehydratases of the inventionreduce the rate of inactivation without undue sacrifice to the rate ofcatalysis. This solution to the problem of dehydratase inactivation ispreferred since it increases the turnover ratio (mol product/molholoenzyme) of coenzyme B₁₂-dependent dehydratase in the microbial host.The effect of increased turnover ratio reduces the dehydratase demand,the coenzyme B₁₂ demand, and the energetic consumption to maintain thatlevel of dehydratase and coenzyme B₁₂. In addition, coenzymeB₁₂-dependent dehydratases have been demonstrated to operate efficientlyfor the production of 1,3-propanediol under industrial processconditions.

In addition to providing a suite of mutant dehydratases, the presentinvention also provides two high throughput assays to facilitatescreening of mutant dehydratases. Both methods rely on the existence ofthe B₁₂-dependent dehydratase in its apoenzyme form in cells that do nothave a source of the coenzyme B₁₂, dehydratase reactivation factor, orB₁₂-dependent dehydratase. Thus, it is possible to precisely control theinitiation of dehydratase activity, based on the addition of coenzymeB₁₂ and substrate glycerol.

Specifically, large libraries of mutated coenzyme B₁₂-dependentdehydratase genes were generated and the gene products expressed andscreened in high throughput fashion for reduced inactivation in thepresence of a glycerol and 1,3-propandiol mixture. The screening methodenabled the facile and rapid identification of those engineered coenzymeB₁₂-dependent dehydratases with reduced inactivation rates and improvedrates of glycerol catalysis. Subsequent rounds of dehydrataseengineering enabled the generation and identification of even moreimproved dehydratase variants.

It will be obvious to one of skill in the art, based on the teachingsherein, that a variety of genes encoding B₁₂-dependent dehydrataseactivity capable of catalyzing the conversion of glycerol to 3-HP shouldbe suitable as a target for mutagenesis and screening, as described inthe present invention. Thus, it will be expected that a variety ofmutant dehydratases having improved reaction kinetics (i.e., anincreased total turnover number in the presence of glycerol and1,3-propanediol) could be identified by means of this invention.

DEFINITIONS

The following abbreviations and definitions are used for theinterpretation of the specification and the claims.

“Polymerase chain reaction” is abbreviated PCR.

“3-Hydroxypropionaldehyde” is abbreviated 3-HP.

The term “dehydratase” is used to refer to any enzyme that is capable ofisomerizing or converting a glycerol molecule to the product3-hydroxypropionaldehyde. As noted above, some dehydratases requirecoenzyme B₁₂ as a cofactor. The terms “coenzyme B₁₂-dependentdehydratase” and “B₁₂-dependent dehydratase” are used interchangeably torefer to those dehydratases which require coenzyme B₁₂. CoenzymeB₁₂-dependent dehydratase comprise the coenzyme B₁₂-dependentdehydratase (E.C. 4.2.1.30) and the coenzyme B₁₂-dependent dehydratase(E.C. 4.2.1.28). Alternatively, dehydratases may be coenzymeB₁₂-independent; these enzymes do not require coenzyme B₁₂ as cofactorand are referred to by the term “B₁₂-independent dehydratase”. For thepurposes of this invention, the term “glycerol dehydratase” are used tospecifically refer to the coenzyme B₁₂-dependent dehydratase (E.C.4.2.1.30) and the term “diol dehydratase” are used to specifically referto the coenzyme B₁₂-dependent dehydratase (E.C. 4.2.1.28). (Applicants'deliberate choice of nomenclature is not to be confused with that ofHartmanis and Stadman, supra and Crous et al., supra who refer toB₁₂-independent dehydratases by the terms “diol dehydratase” and“glycerol dehydratase”, respectively).

The term “apoenzyme” refers to the portion of an enzyme that is composedof protein. An apoenzyme does not include non-protein structures thatmay be required for the enzyme to be functional; and thus, an apoenzymemay be catalytically inactive. The term “cofactor” refers to anon-protein structure that is required by an apoenzyme for catalyticactivity. The term “holoenzyme” refers to the catalytically activeprotein-cofactor complex. A coenzyme B₁₂-dependent dehydratase apoenzymerequires the cofactor coenzyme B₁₂ to form holoenzyme.

The terms “coenzyme B₁₂” and “adenosylcobalamin” are usedinterchangeably to mean 5′-deoxyadenosylcobalamin.

The terms “vitamin B₁₂” and “cyanocobalamin” are used interchangeablyand refer to the derivative of coenzyme B₁₂ where the upper axial5′-deoxy-5′-adenosyl ligand is replaced with a cyano moiety.

“Hydroxocobalamin” refers to a derivative of coenzyme B₁₂ wherein theupper axial 5′-deoxyadenosyl ligand is replaced with a hydroxy moiety.Aquacobalamin is the protonated form of hydroxocobalamin.

Inactivation of a B₁₂-independent dehydratase holoenzyme by glycerol or1,3-propanediol leads to the formation of an inactive cobalamin specieswhich has lost the upper axial 5′-deoxy-5′-adenosyl ligand. The term“coenzyme B₁₂ precursor” refers to a derivation of coenzyme B₁₂ wherethe upper axial 5′-deoxyadenosyl ligand is replaced.

As used herein, the term “GDH” refers specifically to the B₁₂-dependent,glycerol dehydratase isolated from Klebsiella pneumoniae ATCC 25955 andencoded by bp 7044-8711, bp 8724-9308, and bp 9311-9736 of SEQ ID NO:1.The term “GDH” is used to refer to the assembled complex of α, β, andγ-subunits and may refer to apoenzyme or holoenzyme. Reference to anindividual subunit of GDH will specify the subunit, for example“α-subunit of GDH” or “GDH α-subunit”. Similarly, reference may be madeto the amino acid sequence of GDH, referring collectively to the α- andthe β- and the γ-subunits; or, to an individual subunit of GDH. Theamino acid sequences of the α, β, and γ-subunits of GDH are provided asSEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively.

For purposes of this invention disclosure, GDH is used as a referencefor DNA and amino acid sequence as compared to engineered (mutant)derivatives. GDH is also used as a reference for wild-type reactionkinetics against which the reaction kinetics of the engineeredderivatives created by use of the invention are measured. While GDH isused as the reference material herein, any naturally occurring coenzymeB₁₂-dependent dehydratase (e.g., those listed in Table 2) could be usedinterchangeably with GDH in the present invention against which thereaction kinetics of engineered derivatives are measured.

The term “genetically altered” refers to the process of changinghereditary material by transformation or mutation.

As used herein, the term “mutant” refers to a bacterial clone, plasmid,library or vector containing a GDH enzyme or a GDH sequence that hasbeen generated by a process of mutation. Alternatively, the term mutantrefers directly to a GDH enzyme, GDH amino acid sequence, or GDH DNAsequence that has been generated by a process of mutation. Thus, themutant GDH is different than the (wild-type) GDH.

The term “improved reaction kinetics” refers to a reduced rate ofdehydratase inactivation in the presence of glycerol and/or1,3-propanediol, with respect to the wildtype enzyme. Thus, improvedreaction kinetics are related to an increased total enzyme turnovernumber in the presence of glycerol and 1,3-propanediol. This can beachieved by either increasing the k_(cat) and/or decreasing the rate ofenzyme inactivation.

The term “catalytic efficiency” is defined as the k_(cat)/K_(M) of anenzyme. “Catalytic efficiency” is used to quantitate the specificity ofan enzyme for a substrate.

The terms “k_(cat)”, “K_(M)”, and “K_(i)” are known to those skilled inthe art and are described in (Ferst In Enzyme Structure and Mechanism,2^(nd) ed.; W.H. Freeman: New York, 1985; pp 98-120).

The term “k_(cat)” is often called the “turnover number”. The term“k_(cat)” is defined as the maximum number of substrate moleculesconverted to products per active site per unit time, or the number oftimes the enzyme turns over per unit time. k_(cat)=V_(max)/[E], where[E] is the enzyme concentration (Ferst In Enzyme Structure andMechanism, 2^(nd) ed.; W.H. Freeman: New York, 1985; pp 98-120). Theterms “total turnover” and “total turnover number” are used herein torefer to the amount of product formed by the reaction of a B₁₂-dependentdehydratase holoenzyme with glycerol (optionally, in the presence of1,3-propanediol) in the time period between initiation of the reaction(T₀) and that time where complete inactivation of the holoenzyme hasoccurred.

The term “K_(i)” refers to the inhibition constant of 1,3-propanediol.

The term “k_(inact obsd)” refers to the first order rate constant ofinactivation observed in the reaction of a B₁₂-dependent dehydrataseholoenzyme and excess glycerol and/or excess 1,3-propanediol. In thepresence of excess glycerol alone, k_(inact obsd) is equal tok_(inact glycerol). In the presence of excess 1,3-propanediol alone,k_(inact obsd) is equal to k_(inact 1,3-propanediol). In the presence ofboth excess glycerol and excess 1,3-propanediol alone, k_(inact obsd) isequal to a function of both k_(inact glycerol) andk_(inact 1,3-propanediol).

The term “T1” refers to the amount of product made by a GDH enzymereaction measured at 30 sec after the reaction's initiation in thepresence of 10 mM glycerol and 50 mM 1,3-propanediol. T1 is proportionalto k_(cat).

The term “T2” refers to the amount of product made by a GDH enzymereaction measured at 40 min after the reaction's initiation in thepresence of 10 mM glycerol and 50 mM 1,3-propanediol. T2 is proportionalto k_(cat)/k_(inact obsd), reflecting the total enzyme total turnovernumber.

The term “T2/T1 ratio” refers to the ratio of T2 to T1. The T2/T1 ratiois proportional to 1/k_(inact obsd).

The term “T2(600)” refers to the amount of product made by a GDH enzymereaction measured at 40 min after the reaction's initiation in thepresence of 10 mM glycerol and 600 mM 1,3-propanediol. T2(600) isproportional to k_(cat)/k_(inact obsd), reflecting the total enzymetotal turnover number in the presence of 600 mM 1,3-propanediol.

The term “T(600)” refers to the amount of product made by a GDH enzymereaction measured at 70 min after the reaction's initiation in thepresence of 10 mM glycerol and 600 mM 1,3-propanediol. T(600) isproportional to k_(cat)/k_(inact obsd) T(600) value gives a moreaccurate total enzyme turnover number in the presence of 600 mM1,3-propanediol than the T2(600).

The terms “polypeptide” and “protein” are used interchangeably.

The terms “host cell” or “host organism” refer to a microorganismcapable of receiving foreign or heterologous genes and of expressingthose genes to produce an active gene product.

The term “isolated” refers to a protein or DNA sequence that is removedfrom at least one component with which it is naturally associated.

An “isolated nucleic acid molecule” is a polymer of RNA or DNA that issingle- or double-stranded, optionally containing synthetic, non-naturalor altered nucleotide bases. An isolated nucleic acid molecule in theform of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA or synthetic DNA.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, and optionally may include regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” and “wild-type gene” refer to a gene as found innature with its own regulatory sequences. “Chimeric gene” refers to anygene that is not a native gene, comprising regulatory and codingsequences that are not found together in nature. Accordingly, a chimericgene may comprise regulatory sequences and coding sequences that arederived from different sources, or regulatory sequences and codingsequences derived from the same source, but arranged in a mannerdifferent than that found in nature. “Endogenous gene” refers to anative gene in its natural location in the genome of an organism. A“foreign” gene refers to a gene not normally found in the host organism,but that is instead introduced into the host organism by gene transfer.Foreign genes can comprise native genes inserted into a non-nativeorganism, or chimeric genes. A “transgene” is a gene that has beenintroduced into the genome by a transformation procedure.

The terms “encoding” and “coding” refer to the process by which a gene,through the mechanisms of transcription and translation, produces anamino acid sequence. It is understood that the process of encoding aspecific amino acid sequence includes DNA sequences that may involvebase changes that do not cause a change in the encoded amino acid, orwhich involve base changes which may alter one or more amino acids, butdo not affect the functional properties of the protein encoded by theDNA sequence. It is therefore understood that the invention encompassesmore than the specific exemplary sequences.

The term “amino acid” refers to the basic chemical structural unit of aprotein or polypeptide. The amino acids are identified by eitherone-letter code or the three-letter codes for amino acids, in conformitywith the IUPAC-IYUB standards described in Nucleic Acids Research13:3021-3030 (1985) and in the Biochemical Journal 219 (2):345-373(1984), which are herein incorporated by reference.

For a particular protein, point substitution mutations within the DNAcoding region and the resulting amino acid change are specified withreference to a standard DNA and amino acid sequence, using one of thefollowing notations. For example, in the case of mutations in GDH, themutations are described using one of the notations described below:

-   -   1. A detailed notation: First, the nucleotide sequence for the        wild-type codon is presented; followed by an “α”, “β”, or “γ”        symbol (to distinguish mutations in the α-, β-, or γ-subunit of        GDH), the specific wild-type amino acid in three-letter        abbreviation, and its position. The wild-type information is        then followed by the specific nucleotide and amino acid        modification that exist in the referenced mutation. An example        of this notation is: GGG(α-Gly63) to GGA(Gly), wherein the        63^(rd) codon of the α-subunit underwent a silent mutation such        that the nucleotide sequence was altered from “GGG” to “GGA” in        the mutant.    -   2. A “short-hand” notation: An “α”, “β”, or “γ” symbol (to        distinguish mutations in the α- β- or γ-subunit of GDH) is        followed by the wild-type amino acid in one-letter abbreviation,        the codon position, and the one-letter abbreviation for the        mutant amino acid. An example of this notation is: α-V224L,        representing the mutation of the wild-type valine at codon 224        in the α-subunit to leucine in the mutant.        -   It is well known in the art that alterations in a gene which            result in the production of a chemically equivalent amino            acid at a given site (but do not effect the functional            properties of the encoded protein) are common.

“Substantially similar” refers to nucleic acid molecules wherein changesin one or more nucleotide bases result in substitution of one or moreamino acids, but do not affect the functional properties of the proteinencoded by the DNA sequence. “Substantially similar” also refers tonucleic acid molecules wherein changes in one or more nucleotide basesdo not affect the ability of the nucleic acid molecule to mediatealteration of gene expression by antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidmolecules of the instant invention (such as deletion or insertion of oneor more nucleotide bases) that do not substantially affect thefunctional properties of the resulting transcript vis-à-vis the abilityto mediate alteration of gene expression by antisense or co-suppressiontechnology or alteration of the functional properties of the resultingprotein molecule. The invention encompasses more than the specificexemplary sequences.

Each of the proposed modifications is well within the routine skill inthe art, as is determination of retention of biological activity of theencoded products. Moreover, the skilled artisan recognizes thatsubstantially similar sequences encompassed by this invention are alsodefined by their ability to hybridize, under stringent conditions(0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by0.1×SSC, 0.1% SDS), with the sequences exemplified herein. Preferredsubstantially similar nucleic acid fragments of the instant inventionare those nucleic acid fragments whose DNA sequences are at least 80%identical to the DNA sequence of the nucleic acid fragments reportedherein. More preferred nucleic acid fragments are at least 90% identicalto the DNA sequence of the nucleic acid fragments reported herein. Mostpreferred are nucleic acid fragments that are at least 95% identical tothe DNA sequence of the nucleic acid fragments reported herein.

A nucleic acid fragment is “hybridizable” to another nucleic acidfragment, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid fragment can anneal to the other nucleic acidfragment under the appropriate conditions of temperature and solutionionic strength. Hybridization and washing conditions are well known andexemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. MolecularCloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor Laboratory:Cold Spring Harbor, N.Y. (1989), particularly Chapter 11 and Table 11.1therein.

A “substantial portion” refers to an amino acid or nucleotide sequencewhich comprises enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to afford putative identification ofthat polypeptide or gene, either by manual evaluation of the sequence byone skilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Basic Local AlignmentSearch Tool; Altschul et al., J. Mol. Biol. 215:403-410 (1993); see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene-specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid molecule comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence toafford specific identification and/or isolation of a nucleic acidmolecule comprising the sequence. The instant specification teachespartial or complete amino acid and nucleotide sequences encoding one ormore particular proteins. The skilled artisan, having the benefit of thesequences as reported herein, may now use all or a substantial portionof the disclosed sequences for purposes known to those skilled in theart. Accordingly, the instant invention comprises the complete sequencesas reported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

The term “complementary” describes the relationship between doublestranded DNA. For example, with respect to DNA, adenosine iscomplementary to thymine and cytosine is complementary to guanine.Accordingly, the instant invention also includes isolated nucleic acidmolecules that are complementary to the complete sequences as reportedin the accompanying Sequence Listing, as well as those substantiallysimilar nucleic acid sequences.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: 1.) Computational MolecularBiology, Lesk, A. M., Ed.; Oxford University: New York, 1988; 2.)Biocomputing: Informatics and Genome Projects; Smith, D. W., Ed.;Academic: New York, 1993; 3.) Computer Analysis of Sequence Data, PartI; Griffin, A. M. and Griffin, H. G., Eds.; Humana: New Jersey, 1994;4.) Sequence Analysis in Molecular Biology; von Heinje, G., Ed.;Academic: New York, 1987; and 5.) Sequence Analysis Primer, Gribskov, M.and Devereux, J., Eds.; Stockton: New York, 1991. Preferred methods todetermine identity are designed to give the largest match between thesequences tested.

Methods to determine identity and similarity are codified in publiclyavailable computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to: the GCG Pileup program found in the GCG program package,using the Needleman and Wunsch algorithm with their standard defaultvalues of gap creation penalty=12 and gap extension penalty=4 (Devereuxet al., Nucleic Acids Res. 12:387-395 (1984)), BLASTP, BLASTN, and FASTA(Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988)). TheBLASTX program is publicly available from NCBI and other sources (BLASTManual, Altschul et al., Natl. Cent. Biotechnol. Inf., Natl. LibraryMed. (NCBI NLM) NIH, Bethesda, Md.; Altschul et al., J. Mol. Biol.215:403-410 (1990); Altschul et al., Nucleic Acids Res. 25:3389-3402(1997)). Another preferred method to determine percent identity is bythe method of the DNASTAR protein alignment protocol using theJotun-Hein algorithm (Hein et al., Methods Enzymol. 183:626-645 (1990)).Default parameters for the Jotun-Hein method for alignments are: 1.) formultiple alignments: gap penalty=11, gap length penalty=3; and 2.) forpairwise alignments: ktuple=6. As an illustration, by a polynucleotidehaving a nucleotide sequence having at least, for example, 95%“identity” to a reference nucleotide sequence it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence may be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence may be inserted intothe reference sequence. These mutations of the reference sequence mayoccur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among nucleotides in the reference sequence or inone or more contiguous groups within the reference sequence.Analogously, by a polypeptide having an amino acid sequence having atleast, for example, 95% identity to a reference amino acid sequence isintended that the amino acid sequence of the polypeptide is identical tothe reference sequence except that the polypeptide sequence may includeup to five amino acid alterations per each 100 amino acids of thereference amino acid. In other words, to obtain a polypeptide having anamino acid sequence at least 95% identical to a reference amino acidsequence, up to 5% of the amino acid residues in the reference sequencemay be deleted or substituted with another amino acid, or a number ofamino acids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

The term “homologous” refers to a protein or polypeptide native ornaturally occurring in a given host cell. The invention includesmicroorganisms producing homologous proteins via recombinant DNAtechnology.

The term “percent homology” refers to the extent of amino acid sequenceidentity between polypeptides. When a first amino acid sequence isidentical to a second amino acid sequence, then the first and secondamino acid sequences exhibit 100% homology. The homology between any twopolypeptides is a direct function of the total number of matching aminoacids at a given position in either sequence, e.g., if half of the totalnumber of amino acids in either of the two sequences are the same thenthe two sequences are said to exhibit 50% homology.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. The skilled artisan is well aware ofthe “codon-bias” exhibited by a specific host cell in usage ofnucleotide codons to specify a given amino acid.

Modifications to the sequence, such as deletions, insertions, orsubstitutions in the sequence which produce silent changes that do notsubstantially affect the functional properties of the resulting proteinmolecule are also contemplated. For example, alteration in the genesequence which reflect the degeneracy of the genetic code, or whichresult in the production of a chemically equivalent amino acid at agiven site, are contemplated. In some cases, it may in fact be desirableto make mutants of the sequence in order to study the effect ofalteration on the biological activity of the protein. Each of theproposed modifications is well within the routine skill in the art, asis determination of retention of biological activity in the encodedproducts.

The term “expression” refers to the transcription and translation togene product from a gene coding for the sequence of the gene product.

The terms “plasmid”, “vector”, and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitates transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

The term “recombination” will refer to a process whereby geneticcombinations are formed which were not present in parental templatemolecules, by the processes of crossing over or independent assortment.Thus, recombination includes all combinations of genetic sequences thatcan be obtained from the parental template molecules (whereby eachnucleotide position of the newly generated “recombinogenic product(s)”can be derived from any of the parental templates at that particularnucleotide position); and additionally, recombination includes theintroduction of new mutations (i.e., deletions, substitutions,insertions).

The term “recombined polypeptide” means a polypeptide encoded byrecombined genes or DNA. Recombined polypeptides will often have alteredor enhanced properties.

The term “altered properties” as applied to a polypeptide or proteinwill refer to a characteristic, associated with a protein encoded by anucleotide sequence which can be measured by an assay method, where thatcharacteristic is either enhanced or diminished compared with thatassociated with the native sequence. Examples of preferred properties ofan enzyme that may be altered include the enzyme's activity, substratespecificity, stability against inhibitors, thermal stability, proteasestability, solvent stability, detergent stability, and foldingproperties. “Enhanced biological property” refers to an altered propertythat is greater than that associated with the native sequence.“Diminished biological properties” is an altered property that is lessthan that associated with the native sequence.

The term “template(s)” or “parent template(s)” refers to a nucleic acidmolecule that is copied by a DNA or RNA polymerase according to therules of Watson-Crick base pairing to produce a new strand of DNA orRNA. The sequence information in the template (or “model”) is preserved,since the first copy produced from that template molecule has acomplementary sequence. Template molecules may be single ordouble-stranded and derived from any source.

“Replication” is the process in which a complementary copy of a nucleicacid strand of the “template nucleic acid” is synthesized by apolymerase enzyme. In a “primer-directed” replication, this processrequires a hydroxyl group (OH) at the 3′ position of a (deoxy)ribosemoiety of the terminal nucleotide of a “duplexed” “oligonucleotide” toinitiate replication.

The “5′ region” and “3′ region” of a nucleic acid will be used asrelative terms, in reference to the region of nucleotides wherein it isdesirable for recombination to occur. These regions may be within atemplate molecule or within a flanking DNA sequence that is attached tothe template molecules. Unpaired primers will anneal to a portion ofthese 5′ and 3′ regions.

A “flanking sequence” or “flanking DNA fragment” will refer to a shortsegment of DNA that is attached to either the 5′ or 3′ region of atemplate molecule, in order to provide a unique nucleotide sequence(with respect to the template molecule) to which an unpaired primer mayanneal.

A “full length extension product” is a nucleotide sequence produced byprimer-directed replication that has a length very similar (within about100 bases) to that contained between the 5′ and 3′ region of the parenttemplates.

“Amplification” is the process in which replication is repeated incyclic manner such that the number of copies of the “template nucleicacid” is increased in either a linear or logarithmic fashion.

The term “primer” refers to an oligonucleotide (synthetic or occurringnaturally), which is capable of acting as a point of initiation ofnucleic acid synthesis or replication along a complementary strand whenplaced under conditions in which synthesis of a complementary stand iscatalyzed by a polymerase. The primer must have the ability to anneal tothe complementary strand, based on sequence complementarity between theprimer itself and the complementary strand of nucleic acid, however somemismatch in bases is tolerated. Requirements for primer size, basesequence, complementarity and target interaction are discussed ingreater detail below. The term “primer”, as such, is used generallyherein by Applicants to encompass any sequence-binding oligonucleotidewhich functions to initiate the nucleic acid replication process (i.e.,is capable of priming synthesis).

The term “forward primer” will refer to a primer that is capable ofpriming synthesis at the 5′ region on a sense strand of adouble-stranded template molecule or that is capable of primingsynthesis at the 5′ region of a single-stranded template molecule.

The term “reverse primer” will refer to a primer that is capable ofpriming synthesis at the 5′ region on an antisense strand of adouble-stranded template molecule or that is capable of primingsynthesis at the 5′ region of a single-stranded template molecule thatis an antisense strand of a double-stranded template molecule.

The term “paired primers” will refer to a pair of primers, consisting ofa forward and reverse primer, which are designed to anneal to a singletemplate molecule and permit synthesis of an exact copy of that templateby a primer directed nucleic acid amplification process. In the case ofa double-stranded template molecule, the forward and reverse primersenable the synthesis of an exact copy of the double-stranded templatesince the forward primer produces an exact copy of the antisense strand(that is a complementary copy of the sense strand which it is using as atemplate) and the reverse primer produces an exact copy of the sensestrand (that is a complementary copy of the antisense strand which it isusing as a template). In contrast, when the template molecule issingle-stranded, an exact copy of that template is produced using aprimer directed nucleic acid amplification process.

The term “unpaired primers” will refer to a pair of primers, consistingof a forward and reverse primer, which are not designed to anneal to asingle template molecule and permit synthesis of an exact copy of thattemplate by a primer directed nucleic acid amplification process.Instead, the forward primer will anneal to a first template molecule,but will not be able to anneal to a second template molecule. Thereverse primer will anneal to a second template molecule that isdifferent in sequence from the first template molecule, and yet will notbe able to anneal to the first template molecule. This unique design ofunpaired primers ensures that a single- or double-stranded templatemolecule can not be amplified by a primer directed nucleic acidamplification process, unless recombination occurs during replicationvia template switching.

The term “primer directed extension” refers to any method known in theart wherein primers are used to sponsor replication of nucleic acidsequences in the linear or logarithmic amplification of nucleic acidmolecules. For example, primer-directed extension may be accomplished byany of several schemes known in the art including, but not limited to:the polymerase chain reaction (PCR), ligase chain reaction (LCR), andstrand-displacement amplification (SDA).

GDH and the Encoding Genes

With respect to the conversion of glycerol to 3-HP, the dehydrataseresponsible for catalyzing this reaction can be a B₁₂-dependentdehydratase or a B₁₂-independent dehydratase. For the purposes of thisinvention, however, the dehydratase is a B₁₂-dependent dehydratase. ThisB₁₂-dependent dehydratase can be GDH, a glycerol dehydratase (E.C.4.2.1.30), or a diol dehydratase (E.C. 4.2.1.28). Each of these enzymespossess an α₂β₂γ₂ structure. For clarity, due to the wide variation ingene nomenclature used in the literature, a comparative chart showinggene names and GenBank references for dehydratase genes of Klebsiellapneumoniae, Citrobacter freundii, Clostridium pasteurianum, Salmonellatyphimurium, Lactobacillus collinoide, and Klebsiella oxytoca are givenin TABLE 2 to facilitate identification. The genes are also describedin, for example, Daniel et al. (FEMS Microbiol. Rev. 22: 553 (1999)) andToraya and Mori (J. Biol. Chem. 274: 3372 (1999)). TABLE 2 Comparativechart of gene names and GenBank references for dehydratases ORGANISMdehydratase, α dehydratase, β dehydratase, γ (GenBank Reference) gene bpgene bp gene bp K. pneumoniae dhaB1 7044-8711 dhaB2 8724-9308 dhaB39311-9736 (WO 01/012833A2) (SEQ ID NO: 1) K. pneumoniae DhaB1 3047-4714dhaB2 2450-2890 dhaB3 2022-2447 (U30903) K. pneumoniae gldA  121-1788gldB 1801-2385 gldC 2388-2813 (U60992) C. freundii dhaB  8556-10223 dhaC10235-10819 dhaE 10822-11250 (U09771) C. pasteurianum dhaB  84-1748 dhaC1779-2318 dhaE 2333-2773 (AF051373) S. typhimurium pduC 3557-5221 pduD5232-5906 pduE 5921-6442 (AF026270) L. collinoides pduC 2480-4156 pduD4185-4877 pduE 4897-5418 (AJ297723) K. oxytoca pddA  121-1785 pddB1796-2470 pddC 2485-3006 (AF051373)

For the purposes of the present invention, GDH encoded by dhaB1, dhaB2,and dhaB3 (SEQ ID NO:1 and SEQ ID NO: 2, 3, and 4, respectively) of K.pneumoniae (WO 01/012833A2) was used as the target for mutagenesis. Thisenzyme, a glycerol dehydratase, has a preferred substrate of glyceroland is known to consist of two 63 kDa α subunits, two 21 kDa β subunits,and two 16 kDa γ subunits. However, one skilled in the art willrecognize that glycerol dehydratases of Citrobacter freundii,Clostridium pasteurianum, or other K. pneumoniae strains; or, dioldehydratase of Salmonella typhimurium, Klebsiella oxytoca or K.pneumoniae will also be suitable for the techniques described herein.Likewise, any gene(s) encoding a B₁₂-dependent dehydratase activitywherein that activity is capable of catalyzing the conversion ofglycerol to 3-HP should be suitable as a target in the presentinvention, including any amino acid sequence that encompasses amino acidsubstitutions, deletions or additions that do not alter the function ofGDH enzyme. Thus, the skilled person will appreciate that genes encodingGDH isolated from other sources will also be suitable for use in thepresent invention.

B₁₂-dependent Dehydratase Inactivation in the Presence of Glycerol or1,3-Propanediol

B₁₂-dependent dehydratases undergo irreversible inactivation by glycerolduring catalysis or by interaction with 1,3-propanediol. Inactivationinvolves cleavage of the cobalt-carbon (Co—C) bond of the coenzyme B₁₂cofactor, leading to the formation of 5′-deoxyadenosine and an inactivecobalamin species. The inactive cobalamin species remains tightly boundto the dehydratase; dissociation does not occur without the interventionof coenzyme B₁₂-dependent dehydratase reactivation factors.

FIG. 1 illustrates the typical time course associated with GDHinactivation. These enzyme activity assays were conducted using the GDHencoded by dhaB1, dhaB2, and dhaB3 (SEQ ID NO:1 and SEQ ID NO: 2, 3, and4, respectively) of K. pneumoniae (WO 01/012833A2). The upper trace inFIG. 1 shows the time course of the GDH reaction with 10 mM glycerol(K_(m) ˜0.5 mM) as the substrate, while the lower trace shows the effectof including 50 mM 1,3-propanediol (K_(i)˜15 mM) in the assay. Clearly,the rate of 3-HP product formation decreases rapidly with time asinactivation occurs, according to a first-order inactivation rateconstant (k_(inact obsd)).

Mutant GDH with Improved Activities

Based on the observed GDH inactivation, a series of mutant GDHs having areduced inactivation rate, with respect to the wild-type GDH, wascreated. Typically, the approach involves the creating and isolatingmutant enzymes having an increased total turnover number in the presenceof glycerol and 1,3-propanediol. This can be achieved by eitherincreasing the k_(cat) and/or decreasing the rate of enzymeinactivation.

The process of improving GDH activity involves construction of anexpression vector comprising the GDH gene(s), mutagenesis of the GDHcoding sequence, and finally isolation of variants with a decreasedinactivation rate. Subsequent rounds of mutagenesis allow for evolutionof the GDH coding sequence

Mutant B₁₂-dependent dehydratase libraries could be prepared using anywild-type (or substantially similar) B₁₂-dependent dehydratase as thestarting material for mutagenesis.

Traditional Methods of Mutagenesis for B₁₂-Dependent Dehydratase

A variety of approaches may be used for the mutagenesis of theB₁₂-dependent dehydratase. Two suitable approaches used herein includeerror-prone PCR (Leung et al., Techniques, 1:11-15 (1989); Zhou et al.,Nucleic Acids Res. 19:6052-6052 (1991); and Spee et al., Nucleic AcidsRes. 21:777-778 (1993)) and in vivo mutagenesis.

The principal advantage of error-prone PCR is that all mutationsintroduced by this method will be within the B₁₂-dependent dehydratasegene, and any change may be easily controlled by changing the PCRconditions. Alternatively, in vivo mutagenesis may be employed usingcommercially available materials such as the E. coli XL1-Red strain andthe Epicurian coli XL1-Red mutator strain from Stratagene (La Jolla,Calif.; see also Greener and Callahan, Strategies 7:32-34 (1994)). Thisstrain is deficient in three of the primary DNA repair pathways (mutS,mutD and mutT), resulting in a mutation rate 5000-fold higher than thatof wild-type. In vivo mutagenesis does not depend on ligation efficiency(as with error-prone PCR); however, a mutation may occur at any regionof the vector and the mutation rates are generally much lower.

Alternatively, it is contemplated that a mutant B₁₂-dependentdehydratase with reduced inactivation rate may be constructed using themethod of “gene shuffling” (U.S. Pat. No. 5,605,793; U.S. Pat. No.5,811,238; U.S. Pat. No. 5,830,721; and U.S. Pat. No. 5,837,458) or anysimilar means of promoting recombinogenic activity between nucleicacids. The method of gene shuffling is particularly attractive due toits facile implementation and high rate of mutagenesis. The process ofgene shuffling involves the restriction of a gene of interest intofragments of specific size, in the presence of additional populations ofDNA regions of both similarity to (or difference to) the gene ofinterest. This pool of fragments is then denatured and reannealed tocreate a mutated gene. The mutated gene is then screened for alteredactivity.

Wild-type B₁₂-dependent dehydratase sequences may be mutated andscreened for altered or enhanced activity by this method. The sequencesshould be double stranded and can be of various lengths ranging from 50bp to 10 kB. The sequences may be randomly digested into fragmentsranging from about 10 bp to 1000 bp, using restriction endonucleaseswell known in the art (Maniatis, supra). In addition to the full-lengthsequences, populations of fragments that are hybridizable to all (orportions) of the sequence may be added. Similarly, a population offragments which are not hybridizable to the wild-type sequence may alsobe added. Typically these additional fragment populations are added inabout a 10-fold to 20-fold excess by weight as compared to the totalnucleic acid. Generally this process will allow generation of about 100to 1000 different specific nucleic acid fragments in the mixture. Themixed population of random nucleic acid fragments are denatured to formsingle-stranded nucleic acid fragments and then reannealed. Only thosesingle-stranded nucleic acid fragments having regions of homology withother single-stranded nucleic acid fragments will reanneal. The randomnucleic acid fragments may be denatured by heating. One skilled in theart could determine the conditions necessary to completely denature thedouble stranded nucleic acids. Preferably the temperature is from about80° C. to 100° C. The nucleic acid fragments may be reannealed bycooling. Preferably the temperature is from about 20° C. to 75° C.Renaturation can be accelerated by the addition of polyethylene glycol(“PEG”) or salt. The salt concentration is preferably from 0 mM to 200mM. The annealed nucleic acid fragments are next incubated in thepresence of a nucleic acid polymerase and dNTP's (i.e., dATP, dCTP, dGTPand dTTP). The nucleic acid polymerase may be the Klenow fragment, theTaq polymerase or any other DNA polymerase known in the art. Thepolymerase may be added to the random nucleic acid fragments prior toannealing, simultaneously with annealing or after annealing. The cycleof denaturation, renaturation and incubation in the presence ofpolymerase is repeated for a desired number of times. Preferably thecycle is repeated from 2 to 50 times, more preferably the sequence isrepeated from 10 to 40 times. The resulting nucleic acid is a largerdouble-stranded polynucleotide of from about 50 bp to about 100 kB andmay be screened for expression and altered activity by standard cloningand expression protocols (Maniatis, supra).

In addition to the methods exemplified above (which are designed todirectly mutagenize the genes encoding B₁₂-dependent dehydratase),traditional methods of creating mutants could be utilized for thepurposes described herein. For example, wild-type cells havingB₁₂-dependent dehydratase activity may be exposed to a variety of agentssuch as radiation or chemical mutagens and then screened for the desiredphenotype. When creating mutations through radiation either ultraviolet(UV) or ionizing radiation may be used. Suitable short wave UVwavelengths for genetic mutations will fall within the range of 200 nmto 300 nm, where 254 nm is preferred. UV radiation in this wavelengthprincipally causes changes within nucleic acid sequence from guanidineand cytosine to adenine and thymidine. Since all cells have DNA repairmechanisms that would repair most UV induced mutations, agents such ascaffeine and other inhibitors may be added to interrupt the repairprocess and maximize the number of effective mutations. Long wave UVmutations using light in the 300 nm to 400 nm range are also possible;but this range is generally not as effective as the short wave UV light,unless used in conjunction with various activators (such as psoralendyes) that interact with the DNA. Likewise, mutagenesis with chemicalagents is also effective for generating mutants and commonly usedsubstances include chemicals that affect nonreplicating DNA (such asHNO₂ and NH₂OH), as well as agents that affect replicating DNA (such asacridine dyes, notable for causing frameshift mutations). Specificmethods for creating mutants using radiation or chemical agents are welldocumented in the art. See, for example, Thomas D. Brock inBiotechnology: A Textbook of Industrial Microbiology, 2^(nd) ed.;Sinauer Associates: Sunderland, Mass. (1989); or Deshpande, Mukund V.,Appl. Biochem. Biotechnol. 36:227 (1992).

Irrespective of the method of mutagenesis, a gene may be evolved suchthat the enzyme has an increased total turnover number in the presenceof glycerol and 1,3-propanediol. This can be achieved by eitherincreasing the k_(cat) and/or decreasing the rate of enzymeinactivation.

Mutagenesis of B₁₂-Dependent Dehydratase Using a RecombinogenicExtension Method Using Unpaired Primers

FIG. 5 illustrates the principle of the recombinogenic extension methodusing unpaired primers, based on recombination of two genes. This methodrequires that the parent genes have different DNA sequences at their 5′and 3′ ends. If the parent genes have the same 5′ and 3′ sequences, ashort flanking DNA fragment must be attached to the 5′ or 3′ end of thegenes by standard PCR (as shown in Step A of FIG. 5).

Then, the PCR products can be used as templates for the recombinogenicextension method, following the design of two unpaired primers for thethermal cycling. Primer-1 anneals with the 5′ end of template-1, butdoes not bind with the 5′ end of template-2. Primer-2 anneals with the3′ end of template-2, but does not bind to the 3′ end of template-1(Step B). This ensures that neither of the parent templates can beamplified by the thermal cycling reaction.

Short annealing and synthesis cycles are performed, thereby creating aseries of short DNA fragments (Step C). With sufficient homology, someof these DNA fragments will anneal to a different template (i.e.,“template switching”) in a subsequent annealing cycle (Step D).Subsequently, recombinant DNA fragments will be made as shown in FIG. 5.Eventually, recombinant DNA genes with the 5′ end of template-1 and the3′ end of template-2 will be created (Step E).

At this time, previously unpaired primer-1 and primer-2 become pairedprimers for the newly created recombinant genes. Further annealing andsynthesis cycles will amplify the pool of recombinant DNA genes with the5′ end of template-1 and 3′ end of template-2 (Step F of FIG. 5). Duringthe amplification step, the recombinant DNA genes can be furtherrecombined which will increase the number of crossovers of therecombinant DNA genes. Theoretically, all of the amplified productsshould be recombinant DNA products. These recombinant products can bederived directly from the parental template molecules, or additionalmutations (e.g., insertions, deletions, and substitutions) may beincorporated into the final recombinogenic product. The contamination ofthe parent templates in the final reaction mixture is negligible.

Further details concerning this methodology are disclosed in U.S.0/374366, herein incorporated by reference.

Identification of B₁₂-Dependent Dehydrates Variants with ImprovedKinetic Performance

In order to identify (from a large population) those B₁₂-dependentdehydratase variants having improved kinetic performance, development ofa high throughput assay would greatly facilitate screening. A simplescreening method is disclosed herein that relies on two measurements toprovide estimations of improvements in k_(cat) and/or total turnovernumber of a dehydratase variant versus a standard (e.g. wild-type GDH).Specifically, GDH mutant gene libraries were cloned by standard methodsinto E. coli for GDH expression, isolates were obtained and grown inLennox Broth, permeabilized, and then assayed for GDH activity. Whilethe screening method is described for GDH contained in whole cells, theskilled artisan will recognize that the method can be easily modified tobe applicable to enzyme in crude or pure preparations.

The GDH assay developed herein relies on existence of the GDH enzyme inits apoenzyme form in cells that do not have a source of the coenzymeB₁₂, dehydratase reactivation factor, or B₁₂-dependent dehydratase. Thecatalytically active GDH holoenzyme forms only upon addition of coenzymeB₁₂ to the media. Thus, the GDH reaction can effectively be turned “on”with great precision, by addition of coenzyme B₁₂ and substrateglycerol. This enables precise control in duration (timing) of a GDHactivity and, thus, accuracy in GDH activity measurements. The reactionis initiated at time zero (time=t₀) and the product formation during aninitial phase of the reaction (immediately after t₀ and before enzymeinactivation occurs, time=t₁) is determined. Additionally, the productformation is determined for a longer period of time (immediately aftert₀ and after enzyme inactivation is complete, time=t₂). Thedetermination of product (3-HP) formation is based on a colorimetricaldehyde assay (Zurek G., and U. Karst. Analytica Chimica Acta,351:247-257 (1997)).

For GDH in the presence of saturating coenzyme B₁₂ and glycerolconcentrations, the amount of 3-HP (y) produced over time is estimatedbyy=T ₀+AMP(1−exp(−k _(inact obsd)*time)),where T₀ is the amount of 3-HP at t₀ (background), AMP is amount of 3-HPproduced between t₀ and when the GDH is totally inactivated (totalturnover number), and k_(inact obsd) is the observed first orderinactivation rate constant. Since 1,3-propanediol is not present,k_(inact obsd) is due entirely to k_(inact glycerol) (the first orderinactivation rate constant due to glycerol) (see FIG. 1 upper trace.AMP=is equal to [GDH]*k_(cat)/k_(inact obsd). The time course is used toestablish appropriate times (t₁ and t₂) for a fixed concentration ofGDH, coenzyme B₁₂ and glycerol. After correcting for T₀, the amount ofproduct formation at t₁ (T1, before enzyme inactivation occurs) is usedto estimate [GDH]*k_(cat); and, product formation at t₂ (T2, afterenzyme inactivation is complete) is used to estimate the total turnovernumber and [GDH]*k_(cat)/k_(inact obsd). Thus, the T1 value is directlyproportional to k_(cat), the T2 value is directly proportional tok_(cat)/k_(inact obsd), and T2/T1 gives 1/k_(inact obsd).

After appropriate times (t₁ and t₂) are determined for a fixedconcentration of wild-type GDH, coenzyme B₁₂ and glycerol (in theabsence of 1,3-propanediol), T1 and T2 values are redetermined under theexact assay conditions but with the addition of 1,3-propanediol (FIG. 1,lower trace). In the presence of glycerol and 1,3-propanediol,competitive inhibition occurs; and, k_(inact obsd) is a function of bothk_(gly) (the first order inactivation rate constant due to glycerol) andk_(1,3-propanediol) (the first order inactivation rate constant due to1,3-propanediol). At appropriate 1,3-propanediol concentration, the T1value is measurably reduced from the value in the absence of1,3-propanediol (reflecting the competitive inhibition of1,3-propanediol and glycerol) and the T2 value is greatly reduced fromthe value in the absence of 1,3-propanediol (reflecting the fact thatk_(1,3-propanediol)>k_(gly)). For the two-point assay, glycerol ispresent at a concentration of between 5 and 50 mM, preferably 10 mM;and, 1,3-propanediol is present at a concentration of between 10 and 300mM, preferably 50 mM.

Applicants have established conditions under which to examine wild-typeGDH versus mutant variants in the presence of glycerol and1,3-propanediol. On a microscopic level, the introduction of mutationsto GDH may affect k_(cat), the enzyme's affinity for glycerol and/or1,3-propanediol, as well as the respective k_(inact) values. Althoughinitially there was no assurance that the affinities and rate constantswould vary independently of each other, it was useful to consider howvariations in these parameters could affect the results of a two-point(T1 and T2) assay conducted in the presence of 10 mM glycerol and 50 mM1,3-propanediol. Specifically, slow inactivation (a decrease in thek_(inact obsd)) results in an increased T2/T1 ratio. On the other hand,variation in either T1 or T2 could result from changes in the intrinsicproperties of the enzyme. For example, given constant relativeaffinities for glycerol and 1,3-propanediol, if k_(cat) and thek_(inact) values were proportionately reduced, T2/T1 would be increased,but T1 would be low. On the other hand, mutants with normal interactionwith glycerol but with reduced relative affinity for 1,3-propanediol ora lower rate constant for 1,3-propanediol inactivation should show aroughly normal T1, an elevated T2, and a higher T2/T1 ratio. Mutantswith normal glycerol and 1,3-propanediol affinities but with decreasedk_(inact) for either compound would also show normal T1 but increased T2and T2/T1 ratio. An increase in k_(cat) without changes in eitherk_(inact) or in substrate or 1,3-propanediol affinity would increase T1and T2, but not the T2/T1 ratio. Despite the numerous variationsdescribed above, however, the ultimate goal of the work herein is toincrease the total turnover number of the GDH enzyme in the presence ofglycerol and 1,3-propanediol. This can be achieved by either increasingthe k_(cat) and/or decreasing the rate of enzyme inactivation.Accordingly, mutants that exhibited at least one improvement from thegroup consisting of higher T1 and/or higher T2 and/or higher T2/T1values or any combination of those parameters as compared to thewild-type are considered to have improved characteristics.

Measurement of T1 (for the estimation of k_(cat)) is not possible whenglycerol concentration is small compared to 1,3-propanediolconcentration, such that significant inactivation by 1,3-propanedioloccurs. These conditions are relevant because high 1,3-propanediolconcentration and low glycerol concentration are desirable in a processfor the production of 1,3-propanediol. Recognizing this aspect of theproblem, a single point assay is provided as described above except thatglycerol is present at a concentration of between xx and yy mM,preferably 10 mM; and, 1,3-propanediol is present at a concentration ofgreater than 300 mM, preferably 600 mM. In this single point assay, thetime is indicated by t_(end) and the amount of 3-HP produced isindicated by T(XXX) where XXX is the concentration of 1,3-propanediol(mM).

Identification of Critical Amino Acids Affecting Coenzyme B₁₂Inactivation

Applicants disclose a variety of mutant GDH enzymes that have decreasedrates of coenzyme B₁₂ inactivation, as compared to the wild-type gene.These mutants were identified using the methods of mutagenesis andscreening described above. The mutants, the altered amino acid residues,the 1/k_(inact obsd) activity (measured as T2/T1), and T2 are summarizedin Table 1. The SEQ ID NO: of the DNA sequence of the enzyme is providedin the first column of the Table. TABLE 3 SUMMARY OF GDH MUTANTS,CHARACTERIZED BY T2/T1 T2/T1 Strain Mutations ratio T2 Wild-type GDHNone 1.00 1.00 (SEQ ID NO: 1) Xba3007 ACC(γ-Thr53) to GCC(Ala) 4.30 0.78(SEQ ID NO: 40) Xba3029 CTC(α-Leu509) to TTC(Phe) 3.24 0.91 (SEQ ID NO:44) Xba3004 ATG(α-Met306) to CTG(Leu) 2.88 0.63 (SEQ ID NO: 48)ACT(β-Thr45) to GCT(Ala) AAC(β-Asn155) to AGC(Ser) Xba3025 ATC(γ-Ile49)to ACC(Thr) 2.77 0.84 (SEQ ID NO: 52) Xba3038 TTT(α-Phe233) to CTT(Leu)2.24 0.58 (SEQ ID NO: 56) Xba3030 ATG(α-Met257) to GTG(Val) 2.00 0.59(SEQ ID NO: 60) Xba3006 GTG(α-Val44) to GCG(Ala) 1.97 0.51 (SEQ ID NO:64) ACC(α-Thr470) to GCC(Ala) Xba3031 GTC(α-Val226) to GCC(Ala) 1.920.64 (SEQ ID NO: 68) Xba3017 ATC(α-Ile105) to ATT(Ile) 1.90 0.69 (SEQ IDNO: 72) TCA(α-Ser168) to CCA(Pro) Xba3005 ATC(α-Ile67) to GTC(Val) 1.840.72 (SEQ ID NO: 76) GAG(α-Glu209) to GAA(Glu) AAC(β-Asn155) to AAG(Lys)Xba3033 ATG(α-Met257) to ACG(Thr) 1.75 0.72 (SEQ ID NO: 80)GAC(β-Asp181) to GGC(Gly) Xba3032 TAC(α-Tyr70) to AAC(Asn) 1.60 0.91(SEQ ID NO: 84) GTG(α-Val86) to GAG(Glu) Xba3018 TAC(α-Tyr70) toAAC(Asn) 1.60 0.79 (SEQ ID NO: 88) GTT(α-Val74) to GTC(Val) Xba3014CCG(α-Pro430) to TCG(Ser) 1.57 0.66 (SEQ ID NO: 92) GAA(β-Glu25) toGAG(Glu) AAA(γ-Lys27) to AGA(Arg) Xba3024 GTG(α-Val44) to GAG(Glu) 1.440.84 (SEQ ID NO: 96) GTG(α-Val461) to GGG(Gly) Xba3026 ACC(α-Thr350) toGCC(Ala) 1.43 0.93 (SEQ ID NO: 100) Sma3009 ATG(α-Met62) to GTG(Val)2.75 0.71 (SEQ ID NO: 104) ATC(α-Ile63) to GTC(Val) AAA(α-Lys149) toAGA(Arg) Sma3010 ATG(α-Met62) to GTG(Val) 2.03 0.96 (SEQ ID NO: 108)GCG(β-Ala53) to GTG(Val) Sma3014 CAG(α-Gln59) to CGG(Arg) 1.84 0.73 (SEQID NO: 112) ATT(α-Ile314) to GTT(Val) TTT(β-Phe11) to TTA(Leu) Sma3008ATG(α-Met62) to ACG(Thr) 1.75 0.95 (SEQ ID NO: 116) CTG(α-Leu268) toCTA(Leu) Sma3001 AAC(α-Asn520) to AGC(Ser) 1.56 0.97 (SEQ ID NO: 120)PpuMI001 CGG(α-Arg137) to AGG(Arg) 2.43 0.57 (SEQ ID NO: 124)TGC(α-Cys143) to TGT(Cys) CTC(α-Leu148) to CGC(Arg) CCG(α-Pro152) toCCC(Pro) PpuMI002 CGG(α-Arg137) to AGG(Arg) 2.43 0.57 (SEQ ID NO: 128)GAT(α-Asp150) to CAT(His) PpuMI005 CGG(α-Arg137) to AGG(Arg) 1.85 0.81(SEQ ID NO: 132) CAG(α-Gln242) to CAA(Gln) AAA(α-Lys149) to CAA(Gln)CCG(α-Pro152) to CCC(Pro) RsrII001 TTC(α-Phe339) to GTC(Val) 1.92 0.72(SEQ ID NO: 136) CGC(α-Arg346) to CGG(Arg) Sma3002 TAT(α-Tyr271) toTGT(Cys) 2.73 1.60 (SEQ ID NO: 140) TAC(α-Tyr502) to CAC(His) TAA(stopof α) to CAA(Gln) CAA(β-Gln2) to CGA(Arg) TTT(β-Phe11) to TTC(Phe)Sma3003 ATG(α-Met62) to CTG(Leu) 1.95 1.16 (SEQ ID NO: 143) Xba3015GTT(α-Val549) to GCT(Ala) 2.36 1.13 (SEQ ID NO: 147) CTG(β-Leu113) toCCG(Pro) GCC(γ-Ala122) to GTC(Val) GCG(γ-Ala128) to GTG(Val) Xba3008TCT(β-Ser122) to CCC(Pro) 2.12 1.15 (SEQ ID NO: 151) AAA(β-Lys166) toAGA(Arg) Xba3016 ATC(α-Ile102) to ACC(Thr) 1.72 1.18 (SEQ ID NO: 155)Xba3020 CCG(β-Pro152) to ACG(Thr) 1.65 1.04 (SEQ ID NO: 159) Xba3037GAG(α-Glu116) to GAA(Glu) 1.48 1.06 (SEQ ID NO: 163) GTT(α-Val423) toATT(Ile) Xba3036 GGT(α-Gly47) to GGC(Gly) 1.27 1.03 (SEQ ID NO: 167)CGA(α-Arg65) to CAA(Gln) 4BR1001 GGC(α-Gly216)to GGG(Gly) 1.90 1.10 (SEQID NO: 171) GTG(α-Val224) to CTG(Leu) Xba3010 CTG(α-Leu318) to TTG(Leu)1.25 1.39 (SEQ ID NO: 175) AAC(α-Asn447) to AAT(Asn) AAT(α-Asn489) toAGT(Ser) GCC(β-Ala27) to TCC(Ser) Xba3009 ACG(α-Thr77) to GCG(Ala) 0.981.61 (SEQ ID NO: 179) TGC(α-Cys193) to AGC(Ser) TAA(stop of α) toGAA(Glu) AAA(β-Lys56) to AGA(Arg) GCC(β-Ala88) to GCT(Ala) GAT(β-Asp111)to GAA(Glu) CAT(γ-His67) to TAT(Tyr) ACC(γ-Thr114) to TCC(Ser) Xba3023CAC(α-His96) to CAT(His) 1.00 1.22 (SEQ ID NO: 182) ATC(α-Ile102) toGTC(Val) GGG(α-Gly63) to GGA(Gly) 2-F4 GTG(α-Val224) to CTG(Leu) 14.40.5 (SEQ ID NO: 186) TAT(α-Tyr271) to TGT(Cys) TAC(α-Tyr502) to CAC(His)TAA(stop of α) to CAA(Gln) CAA(β-Gln2) to CGA(Arg) TTT(β-Phe11) toTTC(Phe) 12-B1 ACG(α-Thr77) to GCG(Ala) 4.6 1.8 (SEQ ID NO: 189)TGC(α-Cys193) to AGC(Ser) TAA(stop of α) to GAA(Glu) AAA(β-Lys56) toAGA(Arg) GCC(β-Ala88) to GCT(Ala) GAT(β-Asp111) to GAA(Glu) ACC(γ-Thr53)to GCC(Ala) CAT(γ-His67) to TAT(Tyr) ACC(γ-Thr114) to TCC(Ser) 13-B7TAT(α-Tyr271) to TGT(Cys) 2.4 0.8 (SEQ ID NO: 192) TAC(α-Tyr502) toCAC(His) TAA(stop of α) to CAA(Gln) CAA(β-Gln2) to CGA(Arg) TTT(β-Phe11)to TTC(Phe) ACC(γ-Thr53) to GCC(Ala) 16-H5 TAT(α-Tyr271)to TGT(Cys) 16.81.2 (SEQ ID NO: 195) TAC(α-Tyr502) to CAC(His) CTC(α-Leu509) to TTC(Phe)TAA(stop of α) to CAA(Gln) CAA(β-Gln2) to CGA(Arg) TTT(β-Phe11) toTTC(Phe) 1-E1 TAA(stop of α) to CAA(Gln) 0.72 1.82 (SEQ ID NO: 198)22-G7 TAA(stop of α) to GAA(Glu) 0.75 1.79 (SEQ ID NO: 201) 7A-C1TAT(α-Tyr271) to TGT(Cys) 1.01 0.43 (SEQ ID NO: 204) 7C-A5 CAA(β-Gln2)to CGA(Arg) 1.02 0.95 (SEQ ID NO: 208) 8-C9 TAC(α-Tyr502) to CAC(His)1.3 1.83 (SEQ ID NO: 212) TAA(stop of α) to CAA(Gln) CAA(β-Gln2) toCGA(Arg) TTT(β-Phe11) to TTC(Phe) 9-D7 TAT(α-Tyr271) to TGT(Cys) 0.981.57 (SEQ ID NO: 215) TAA(stop of α) to CAA(Gln) CAA(β-Gln2) to CGA(Arg)TTT(β-Phe11) to TTC(Phe) 10-G6 TAT(α-Tyr271) to TGT(Cys) 2.56 1.55 (SEQID NO: 218) TAC(α-Tyr502) to CAC(His) TAA(stop of α) to CAA(Gln)TTT(β-Phe11) to TTC(Phe) 21-D10 TAA(stop of α) to CAA(Gln) 3.85 1.91(SEQ ID NO: 221) ACC(γ-Thr53) to GCC(Ala) 20-B9 CTC(α-Leu509) toTTC(Phe) 3.86 1.95 (SEQ ID NO: 225) TAA(stop of α) to CAA(Gln) 18-D7TAC(α-Tyr502) to CAC(His) 5.38 1.30 (SEQ ID NO: 229) TAA(stop of α) toCAA(Gln) CAA(β-Gln2) to CGA(Arg) TTT(β-Phe11) to TTC(Phe) ACC(γ-Thr53)to GCC(Ala) 17-F6 TAC(α-Tyr502) to CAC(His) 3.63 1.77 (SEQ ID NO: 233)CTC(α-Leu509) to TTC(Phe) TAA(stop of α) to CAA(Gln) CAA(β-Gln2) toCGA(Arg) TTT(β-Phe11) to TTC(Phe) 15-E4 TAC(α-Tyr502) to CAC(His) 3.961.66 (SEQ ID NO: 237) TAA(stop of α) to CAA(Gln) CAA(β-Gln2) to CGA(Arg)TTT(β-Phe11) to TTC(Phe) GTG(α-Val224) to CTG(Leu) KG002 CTG(β-Leu113)to CCG(Pro) 1.03 1.22 (SEQ ID NO: 241) ACC(γ-Thr114) to GCC(Ala)GCT(α-Ala309) to GCC(Ala) AAC(α-Asn468) to AAT(Asn) KG003 GTG(α-Val224)to ATG(Met) 1.05 0.92 (SEQ ID NO: 245) CCG(α-Pro450) to CCA(Pro) KG004GTC(α-Val226) to GCC(Ala) 1.53 1.14 (SEQ ID NO: 249) ATG(α-Met306) toTTG(Leu) KG005 ACT(α-Asn288) to ACC(Asn) 1.62 1.33 (SEQ ID NO: 253)ATG(α-Met306) to TTG(Leu) CCG(β-Pro152) to TCG(Ser) KG006 ATG(α-Met62)to ACG(Thr) 1.09 1.22 (SEQ ID NO: 257) KG007 GTC(α-Val115) to GCC(Ala)1.11 1.16 (SEQ ID NO: 261) CTG(β-Leu13) to CCG(Pro) KG010 AAT(α-Asn151)to AAC(Asn) 1.15 1.06 (SEQ ID NO: 265) TTC(α-Phe513) to CTC(Leu) KG011ATG(α-Met214) to TTG(Leu) 1.35 1.03 (SEQ ID NO: 269) GCG(α-Ala460) toGCA(Ala) GAA(α-Glu462) to GAG(Glu) ACC(α-Thr499) to GCC(Ala) GAT(β-Asp24to GGT(Gly) GAA(β-Glu29) to GAG(Glu) CTG(β-Leu58) to CTT(Leu)CGG(β-Arg70) to CGA(Arg) GAG(β-Glu130) to GGG(Gly) AAA(γ-Lys4) toAAG(Lys) KG012 TTA(α-Leu217) to GTA(Val) 1.15 1.01 (SEQ ID NO: 273)KG014 ATG(α-Met62) to GTGVal) 1.10 0.95 (SEQ ID NO: 277) GAT(β-Asp24) toGAA(Glu) KG016 CGG(α-Arg137) to AGG(Arg) 0.97 1.02 (SEQ ID NO: 281)AAC(α-Asn141) to ATC(Ile) GAT(α-Asp150) to GAC(Asp) GCG(α-Ala231) toACG(Thr) GGC(αGly236) to AGC(Ser) KG017 TCA(α-Ser41) to TCG(Ser) 0.931.05 (SEQ ID NO: 285) GCG(α-Ala119) to ACG(Thr) AAC(α-Asn447) toAAT(Asn) KG021 GTC(α-Val226) to GCC(Ala) 1.29 0.93 (SEQ ID NO: 289)KG023 CCG(β-Pro152) to TCG(Ser) 1.21 0.88 (SEQ ID NO: 293) KG001AGC(α-Ser219) to AAC(Asn) 5.7 0.80 (SEQ ID NO: 297)

Additional mutants, also identified using the methods of mutagenesis andscreening described above, are summarized below in Table 4. This tablepresents information concerning each mutant, the altered amino acidresidues, and the T(600) activity. The SEQ ID NO: of the DNA sequence ofthe enzyme is provided in the first column of the Table. TABLE 4 SUMMARYOF GDH MUTANTS, CHARACTERIZED BY T(600) Strain Mutations T₍₆₀₀₎Wild-type GDH — 1 GDH-SM1-G11 TAA(stop of α) to CAA(Gln) 4.3 (SEQ ID NO:301) ACC(γ-Thr53) to TCC(Ser) GDH-SM2-B11 TAA(stop of α) to CAA(Gln) 4.1(SEQ ID NO: 304) CTC(α-Leu509) to TTT(Phe) GDH-SM3-D2 GTG(α-Val224) toTTG(Leu) 4.0 (SEQ ID NO: 307) TAC(α-Tyr502) to CAC(His) TAA(stop of α)to CAA(Gln) CAA(β-Gln2) to CGA(Arg) TTT(β-Phe11) to TTC(Phe) GDH-SM4-H2TAA(stop of α) to CAA(Gln) 4.1 (SEQ ID NO: 310) ACC(γ-Thr53) to TGT(Cys)SHGDH12 GTT(α-Val74) to ATT(Ile) 5.9 (SEQ ID NO: 319) GTG(α-Val224) toTTG(Leu) CGC(α-Arg425) to CGT(Arg) TAC(α-Tyr502) to CAC(His) TAA(stop ofα) to CAA(Gln) CAA(β-Gln2) to CGA(Arg) TTT(β-Phe11) to TTC(Phe)AAA(β-Lys14) to AGA(Arg) SHGDH22 GGC(α-Gly216) to GGG(Gly) 5.8 (SEQ IDNO: 322) GTG(α-Val224) to TTG(Leu) CAG(α-Gln337) to CAA(Gln)CGC(α-Arg533) to GGC(Gly) ACC(α-Thr553) to ACG(Thr) TAA(stop of α) toCAA(Gln) ATC(γ-Ile21) to ACC(Thr) CTG(γ-Leu137) to CTA(Leu) SHGDH24CGT(α-Arg134) to CGC(Arg) 5.6 (SEQ ID NO: 328) GGC(α-Gly216) to GGG(Gly)GTG(α-Val224) to TTG(Leu) AGC(α-Ser481) to AGT(Ser) ACC(α-Thr553) toACG(Thr) TAA(stop of α) to CAA(Gln) SHGDH25 ATG(α-Met62) to CTG(Leu) 5.1(SEQ ID NO: 334) GTG(α-Val124) to GCG(Ala) GGC(α-Gly216) to GGG(Gly)GTG(α-Val224) to TTG(Leu) TAA(stop of α) to CAA(Gln) SHGDH29GCC(α-Ala376) to GCT(Ala) 4.6 (SEQ ID NO: 337) CTC(α-Leu509) to TTT(Phe)ACC(α-Thr553) to ACG(Thr) TAA(stop of α) to CAA(Gln) CAG(γ-Gln101) toCGG(Arg) SHGDH37 GTG(α-Val224) to TTG(Leu) 6.6 (SEQ ID NO: 313)TAC(α-Tyr502) to CAC(His) TAA(stop of α) to CAA(Gln) CAA(β-Gln2) toCGA(Arg) TTT(β-Phe11) to TTC(Phe) GAG(γ-Glu35) to AAG(Lys) SHGDH38GTG(α-Val224) to TTG(Leu) 5.7 (SEQ ID NO: 325) TAC(α-Tyr502) to CAC(His)TAA(stop of α) to CAA(Gln) CAA(β-Gln2) to CGA(Arg) TTT(β-Phe11) toTTC(Phe) GGG(β-Gly19) to GAG(Glu) GAA(β-Glu64) to GAG(Glu) CTT(β-Leu67)to CTC(Leu) AAT(γ-Asn72) to AGT(Ser) SHGDH43 GGC(α-Gly216) to GGG(Gly)5.6 (SEQ ID NO: 331) GTG(α-Val224) to TTG(Leu) GAG(α-Glu240) to GAA(Glu)GTG(α-Val301) to GTA(Val) ACC(α-Thr553) to ACG(Thr) TAA(stop of α) toCAA(Gln) AAA(β-Lys166) to AGA(Arg) AAA(β-Lys173) to GAA(Glu)ACC(γ-Thr53) to TCC(Ser) SHGDH51 TTC(α-Phe339) to GTC(Val) 6.2 (SEQ IDNO: 316) CGC(α-Arg346) to CGG(Arg) ACC(α-Thr553) to ACG(Thr) TAA(stop ofα) to CAA(Gln) CCC(β-Pro184) to CCT(Pro) ACC(γ-Thr53) to GCC(Ala)

A variety of mutant GDH enzymes are disclosed above having decreasedrates of coenzyme B₁₂ inactivation. Preferred mutants of the so presentinvention include: SEQ ID NOs: 40, 44, 48, 52, 56, 60, 64, 68, 72, 76,80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136,140, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 182, 186, 189,192, 195, 198, 201, 204, 208, 212, 215, 218, 221, 225, 229, 233, 237,241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293,297, 301, 304, 307, 310, 313, 316, 319, 322, 325, 328, 331, 334, and337. More preferred mutants of the present invention are SEQ ID NOs:140, 179, 186, 189, 192, 195, 198, 201, 212, 215, 218, 301, 304, 307,310, 313, 316, 319, 322, 325, 328, 331, 334, and 337. Most preferredmutants of the present invention are SEQ ID NOs: 313, 322, and 328.

In addition to the mutants above, a large pool of mutants havingimproved reaction kinetics could also be synthesized, each furtherhaving some combination of the mutations listed in Tables 2 and 3. Forexample, each mutation in a mutant GDH having multiple-point mutationscan be evaluated for desirable effects toward improving the overallreaction kinetics of the enzyme. Any mutations that did not enhance thereaction kinetics could be returned to the wild-type sequence, ifdesired. Likewise, a GDH mutant from the list above having only asingle-point mutation could be combined with another mutation disclosedabove, to further improve the enzyme's activity. Applicants disclose thefollowing useful mutations in a GDH enzyme, which may be used in avariety of combinations to produce alternative mutant GDH enzymes thathave decreased rates of coenzyme B₁₂ inactivation, as compared to thewild-type gene. These mutations include:

1. In the α-subunit: TCA(α-Ser41) to TCG(Ser); GTG(α-Val44) to GCG(Ala);GTG(α-Val44) to GAG(Glu); GGT(α-Gly47) to GGC(Gly); CAG(α-Gln59) toCGG(Arg); ATG(α-Met62) to GTG(Val); ATG(α-Met62) to ACG(Thr);ATG(α-Met62) to CTG(Leu); ATC(α-Ile63) to GTC(Val); GGG(α-Gly63) toGGA(Gly); CGA(α-Arg65) to CAA(Gln); ATC(α-Ile67) to GTC(Val);TAC(α-Tyr70) to AAC(Asn); GTT(α-Val74) to GTC(Val); GTT(α-Val74) toATT(Ile); ACG(α-Thr77) to GCG(Ala); GTG(α-Val86) to GAG(Glu);CAC(α-His96) to CAT(His); ATC(α-Ile102) to ACC(Thr); ATC(α-Ile102) toGTC(Val); ATC(α-Ile105) to ATT(Ile); GTC(α-Val115) to GCC(Ala);GAG(α-Glu 116) to GAA(Glu); GCG(α-Ala119) to ACG(Thr); GTG(α-Val124) toGCG(Ala); CGT(α-Arg134) to CGC(Arg); CGG(α-Arg137) to AGG(Arg);AAC(α-Asn141) to ATC(Ile); TGC(α-Cys143) to TGT(Cys); CTC(α-Leu148) toCGC(Arg); AAA(α-Lys149) to AGA(Arg); AAA(α-Lys149) to CAA(Gln);GAT(α-Asp150) to CAT(His); GAT(α-Asp150) to GAC(Asp); AAT(α-Asn151) toAAC(Asn); CCG(α-Pro152) to CCC(Pro); TCA(α-Ser168) to CCA(Pro);TGC(α-Cys193) to AGC(Ser); GAG(α-Glu209) to GAA(Glu); ATG(α-Met214) toTTG(Leu); GGC(α-Gly216) to GGG(Gly); TTA(α-Leu217) to GTA(Val);AGC(α-Ser219) to AAC(Asn); GTG(α-Val224) to CTG(Leu); GTG(α-Val224) toATG(Met); GTG(α-Val224) to TTG(Leu); GTC(α-Val226) to GCC(Ala);GCG(α-Ala231) to ACG(Thr); TTT(α-Phe233) to CTT(Leu); GGC(αGly236) toAGC(Ser); GAG(α-Glu240) to GAA(Glu); CAG(α-Gln242) to CAA(Gln);ATG(α-Met257) to GTG(Val); ATG(α-Met257) to ACG(Thr); CTG(α-Leu268) toCTA(Leu); TAT(α-Tyr271) to TGT(Cys); ACT(α-Asn288) to ACC(Asn);GTG(α-Val301) to GTA(Val); ATG(α-Met306) to CTG(Leu); ATG(α-Met306) toTTG(Leu); GCT(α-Ala309) to GCC(Ala); ATT(α-Ile314) to GTT(Val);CTG(α-Leu318) to TTG(Leu); CAG(α-Gln337) to CAA(Gln); TTC(α-Phe339) toGTC(Val); CGC(α-Arg346) to CGG(Arg); ACC(α-Thr350) to GCC(Ala);GCC(α-Ala376) to GCT(Ala); GTT(α-Val423) to ATT(Ile); CGC(α-Arg425) toCGT(Arg); CCG(α-Pro430) to TCG(Ser); AAC(α-Asn447) to AAT(Asn);CCG(α-Pro450) to CCA(Pro); GCG(α-Ala460) to GCA(Ala); GTG(α-Val461) toGGG(Gly); GAA(α-Glu462) to GAG(Glu); AAC(α-Asn468) to AAT(Asn);ACC(α-Thr470) to GCC(Ala); AGC(α-Ser481) to AGT(Ser); AAT(α-Asn489) toAGT(Ser); ACC(α-Thr499) to GCC(Ala); TAC(α-Tyr502) to CAC(His);CTC(α-Leu509) to TTC(Phe); CTC(α-Leu509) to TTT(Phe); TTC(α-Phe513) toCTC(Leu); AAC(α-Asn520) to AGC(Ser); CGC(α-Arg533) to GGC(Gly);GTT(α-Val549) to GCT(Ala); ACC(α-Thr553) to ACG(Thr); TAA(stop of α) toCAA(Gln); TAA(stop of α) to GAA(Glu);

2. In the β-subunit: CAA(β-Gln2) to CGA(Arg); TTT(β-Phe11) to TTA(Leu);TTT(β-Phe11) to TTC(Phe); CTG(β-Leu13) to CCG(Pro); AAA(β-Lys14) toAGA(Arg); GGG(β-Gly19) to GAG(Glu); GAT(β-Asp24 to GGT(Gly);GAT(β-Asp24) to GAA(Glu); GAA(β-Glu25) to GAG(Glu); GCC(β-Ala27) toTCC(Ser); GAA(β-Glu29) to GAG(Glu); ACT(β-Thr45) to GCT(Ala);GCG(β-Ala53) to GTG(Val); AAA(β-Lys56) to AGA(Arg); CTG(β-Leu58) toCTT(Leu); GAA(β-Glu64) to GAG(Glu); CTT(β-Leu67) to CTC(Leu);CGG(β-Arg70) to CGA(Arg); GCC(β-Ala88) to GCT(Ala); GAT(β-Asp111) toGAA(Glu); CTG(β-Leu113) to CCG(Pro); TCT(β-Ser122) to CCC(Pro);GAG(β-Glu130) to GGG(Gly); CCG(β-Pro152) to ACG(Thr); CCG(β-Pro152) toTCG(Ser); AAC(β-Asn155) to AGC(Ser); AAC(β-Asn155) to AAG(Lys);AAA(β-Lys166) to AGA(Arg); AAA(β-Lys173) to GAA(Glu); GAC(β-Asp181) toGGC(Gly); CCC(β-Pro184) to CCT(Pro); and

3. In the γ-subunit: AAA(γ-Lys4) to AAG(Lys); ATC(γ-Ile21) to ACC(Thr);AAA(γ-Lys27) to AGA(Arg); GAG(γ-Glu35) to AAG(Lys); ATC(γ-Ile49) toACC(Thr); ACC(γ-Thr53) to GCC(Ala); ACC(γ-Thr53) to TCC(Ser);ACC(γ-Thr53) to TGT(Cys); CAT(γ-His67) to TAT(Tyr); AAT(γ-Asn72) toAGT(Ser); CAG(γ-Gln101) to CGG(Arg); ACC(γ-Thr114) to TCC(Ser);ACC(γ-Thr114) to GCC(Ala); GCC(γ-Ala122) to GTC(Val); GCG(γ-Ala128) toGTG(Val); and CTG(γ-Leu137) to CTA(Leu).

Similar nucleotide and amino acid mutations could be made in otherB₁₂-dependent dehydratases other than wild-type GDH. Aligning theB₁₂-dependent dehydratase of interest with GDH would indicate the exactnucleotide/base position that required mutation.

The invention encompasses not only the specific mutations describedabove, but also those that allow for the substitution of chemicallyequivalent amino acids. So, for example, where a substitution of anamino acid with the aliphatic, nonpolar amino acid alanine is made, itwill be expected that the same site may be substituted with thechemically equivalent amino acid serine.

Protein Engineering of Dehydratases

It is now possible to attempt to modify many properties of proteins bycombining information on three-dimensional structure and classicalprotein chemistry with methods of genetic engineering and moleculargraphics, i.e. protein engineering. This approach to obtaining enzymeswith altered activities relies first on the generation of a modelmolecule, or the use of a known structure that has a similar sequence toan unknown structure.

For the purposes herein, the 3-dimensional crystal structure ofsubstrate-free form B₁₂-dependent glycerol dehydratase has beenpreviously determined by X-ray crystallography (Liao et al., J.Inorganic Biochem. (in press)); additionally, the structure of theenzyme in complex with 1,2-propanediol has also been reported (Yamanishiet al., Eur. J. Biochem. 269:4484-4494 (2002)). With these 3-dimensionalmodels of B₁₂-dependent glycerol dehydratases, and an understanding ofwhere “hot spots” of mutations are typically located within thosedehydratase enzymes showing improved activity in reaction kinetics (suchthat the rate of suicide inactivation is reduced), one can targetregions within the dehydratase structure where alternative modificationsin structure might bring about desired changes in the properties of theprotein. Thus, for example, regional site-directed mutagenesis targetedtoward the following wild type residues would be expected to yieldadditional dehydratase mutants with improved catalytic activities:

-   -   1.) Residues 62-70 (encompassing the second α helix from the        N-terminal of the α-subunit); and/or    -   2.) Residues 219-236 (a region in the vicinity of the active        site, encompassing a portion of the fourth β-strand of the TIM        barrel and the following loop and a short helix of the        α-subunit).        Expression of a Recombinant Dehydrates in a Host Cell

Suitable host cells for the recombinant production of 3-HP and1,3-propanediol by the expression of a gene encoding a dehydratase maybe either prokaryotic or eukaryotic and will be limited only by theirability to express active enzymes. Preferred hosts will be thosetypically useful for production of 3-HP or 1,3-propanediol such asCitrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter,Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces,Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula,Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella,Bacillus, Streptomyces and Pseudomonas. More preferred in the presentinvention are Escherichia coli, E. blattae, Klebsiella, Citrobacter, andAerobacter.

Vectors, methods of transformation, and expression cassettes suitablefor the cloning, transformation and expression of genes encoding asuitable dehydratase into a host cell will be well known to one skilledin the art. Suitable vectors are those which are compatible with thebacterium used as a host cell. Thus, suitable vectors can be derived,for example, from a bacteria, a virus (e.g., bacteriophage T7 or a M-13derived phage), a cosmid, a yeast, or a plant. Protocols for obtainingand using such vectors are known to those individuals in the art(Sambrook et al., supra).

Typically, the vector or cassette contains sequences directingtranscription and translation of the relevant gene(s), a selectablemarker, and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene thatharbors transcriptional initiation controls and a region 3′ of the DNAfragment that controls transcriptional termination. It is most preferredwhen both control regions are derived from genes homologous to thetransformed host cell although it is to be understood that such controlregions need not be derived from the genes native to the specificspecies chosen as a production host.

Initiation control regions (or promoters) useful to drive expression ofthe relevant genes of the present invention in the desired host cell arenumerous and familiar to those skilled in the art. Virtually anypromoter capable of driving these genes is suitable for the presentinvention including, but not limited to: CYC1, HIS3, GAL1, GAL10, ADH1,PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful forexpression in Saccharomyces); AOX1 (useful for expression in Pichia);and lac, trp, λP_(L), λP_(R), T7, tac, and trc (useful for expression inE. coli).

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary; however, it is most preferred if included.

Once suitable cassettes are constructed they are used to transformappropriate host cells. Introduction of the cassette containing thegenes encoding a mutant dehydratase into a host cell may be accomplishedby known procedures such as by transformation (e.g., usingcalcium-permeabilized cells, electroporation), or by transfection usinga recombinant phage virus (Sambrook et al., supra).

Industrial Production via Fermentation

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates are well known to one of skill in theart of fermentation science. Preferred carbon substrates are glycerol,dihydroxyacetone, monosaccharides, oligosaccharides, polysaccharides,and one-carbon substrates. More preferred are sugars (e.g., glucose,fructose, sucrose) and single carbon substrates (e.g., methanol andcarbon dioxide). Most preferred is glucose.

In addition to an appropriate carbon source, fermentation media mustcontain suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for the growthof the cultures and promotion of the enzymatic pathway necessary for3-HP and 1,3-propanediol production. Particular attention is given toCo(II) salts and/or vitamin B₁₂ or precursors thereof.

Typically, cells are grown at 30° C. in appropriate media. Preferredgrowth media in the present invention are common commercially preparedmedia such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth orYeast Malt Extract (YM) broth. Other defined or synthetic growth mediamay also be used and the appropriate medium for growth of the particularmicroorganism will be known by someone skilled in the art ofmicrobiology or fermentation science.

It is contemplated that the present invention may be practiced usingbatch, fed-batch or continuous processes and that any known mode offermentation would be suitable (a variety of methods are detailed byBrock, supra). Additionally, it is contemplated that cells may beimmobilized on a substrate as whole cell catalysts and subjected tofermentation conditions for 3-HP or 1,3-propanediol production.

It is expected that improved B₁₂-dependent dehydratase mutants will beuseful for the production of 3-HP and 1,3-propanediol in a variety ofprocesses, independent of the carbon substrate used (U.S. Pat. No.5,686,276, glycerol to 3-HPA). Recombinants of the relevant strainscould be swapped out of the dehydratase described in the plasmids ofU.S. Ser. No. 10/420,587 (DuPont 2002), which is incorporated herein byreference.

The present invention is further defined in the following EXAMPLES. Itshould be understood that these EXAMPLES, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these EXAMPLES, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

EXAMPLES General Methods

Procedures required for PCR amplification, DNA modifications by endo-and exonucleases (for generating desired ends for cloning of DNA andligation), and bacterial transformation are well known in the art.Standard molecular cloning techniques are used herein and are describedby Sambrook, J., Fritsch, E. F. and Maniatis, T. in Molecular Cloning: ALaboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory: Cold SpringHarbor, N.Y., 1989; hereinafter “Maniatis”); by Silhavy, T. J., Bennan,M. L. and Enquist, L. W. in Experiments with Gene Fusions (Cold SpringHarbor Laboratory: Cold Spring, N.Y., 1984); and by Ausubel et al. inCurrent Protocols in Molecular Biology (Greene Publishing andWiley-Interscience; 1987).

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in: 1.) Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, Eds., American Society for Microbiology: Washington,D.C., 1994); or 2.) by Brock, T. D. in Biotechnology: A Textbook ofIndustrial Microbiology, 2nd ed. (Sinauer Associates: Sunderland, Mass.,1989). All reagents, restriction enzymes and materials used for thegrowth and maintenance of bacterial cells were obtained from AldrichChemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.),GIBCO/BRL (Gaithersburg, Md.), Sigma Chemical Company (St. Louis, Mo.),or Promega (Madison, Wis.), unless otherwise specified. PCR reactionswere run on GeneAMP PCR System 9700 using Amplitaq or Amplitaq Goldenzymes (PE Applied Biosystems, Foster City, Calif.), unless otherwisespecified. The cycling conditions and reactions were standardizedaccording to the manufacturers' instructions, unless otherwise specifiedherein.

DNA sequencing reactions were performed on an ABI 377 automatedsequencer (PE Applied Biosystems) or on a PTC-200 DNA Engine (MJResearch, Waltham, Mass.) using the Expand High Fidelity PCR System(Roche Applied Science, Indianapolis, Ind.), unless otherwise specified.Likewise, data was managed using the Vector NTI program (InforMax, Inc.,Bethesda, Md.) or DNAstar program (DNASTAR Inc., Madison, Wis.).

The meaning of abbreviations is as follows: The meaning of abbreviationsis as follows: “sec” means second(s), “min” means minute(s), “hr” meanshour(s), “d” means day(s), “μL” means microliter(s), “mL” meansmilliliter(s), “L” means liter(s), “mm” means millimeter(s), “μm” meansmicrometer(s), “nm” means nanometer(s), “mM” means millimolar, “μM”means micromolar, “nM” means nanomolar, “M” means molar, “mmol” meansmillimole(s), “μmol” mean micromole(s), “ng” means nanogram, “mg” meansmilligram(s), “g” means gram(s), “kB” means kilobase(s), “mU” meansmilliunit(s), and “U” means unit(s).

Strains, Vectors and Culture Conditions

Escherichia coli BL21 (DE3) cells were used for enzyme over-expression(Shuster, B. and Retey, J., FEBS Lett. 349:252-254 (1994)). Escherichiacoli XL1-Blue cells were purchased from Stratagene (La Jolla, Calif.).Wild-type Escherichia coli 5K was originally obtained from Coli GeneticStock Center (CGSC #4510; Yale University, New Haven, Conn.) andlysogenized with lambda DE3 (5K(DE3)). Vector pBluescript II SK+ waspurchased from Stratagene.

All kits for molecular biological applications were used according tothe manufacturers' instructions, unless otherwise specified.

Example 1 Construction of the “Xba-Library” Random Mutagenesis Targetingthe α-, β- and γ-Subunits of GDH

A random mutant library targeting the Klebsiella pneumonia dhaB1, dhaB2,and dhaB3 genes was created, using error-prone PCR amplification.Representative sequence analysis of the library demonstrated that therewere approximately 4.2 point mutations per kB; enzyme activitymeasurements determined that about 15-25% of the mutants in the librarywere active.

Error-Prone PCR Amplification

Emptage et al. (WO01/12833) describes the construction of plasmid pDT2,which comprises the Klebsiella pneumonia dhaB1, dhaB2, and dhaB3 genes(SEQ ID NO:1). The plasmid pGD20 was constructed by inserting theHindIII/XbaI fragment of pDT2 (containing dhaB1, dhaB2, and dhaB3) intopBluescript II SK+ (Stratagene), which places expression of the GDHgenes under the control of the T7 promoter.

A randomly generated mutant library targeting all three genes of GDH wascreated. First, the sequence comprising dhaB1 (1668 bp), dhaB2 (585 bp)and dhaB3 (426 bp) was amplified from pGD20 by error-prone PCR using thefollowing primers: DHA-F1 (SEQ ID NO:5) and DHA-R1 (SEQ ID NO:6). AClontech mutagenesis kit (Clontech Laboratories, Inc., Palo Alto,Calif.) was used for performing error-prone PCR. The reaction mixtureconsisted of the following: 38 μl PCR grade water, 5 μl 10× AdvanTaqPlus Buffer, 2 μl MnSO₄ (8 mM), 1 μl dGTP (2 mM), 1 μl 50× DiversifydNTP Mix, 1 μl Primer mix, 1 μl template DNA, and 1 μl AdvanTaq PlusPolymerase. The thermal cycling reaction was carried out according tothe manufacturers' instructions. The 2.7 kB PCR products were digestedwith Hind III/Xba I, and prepared for ligation.

Mutant Library Construction

Although the entire insert containing all three genes of GDH can beremoved from the pGD20 construct using a Hind III and Xba I digestion,the insert size is approximately 2.7 kB, while the vector size is about2.9 kB. To facilitate separation of these two fragments on an agarosegel, pGD20 was digested using Hind III, Xba I and Pst I. Pst I does notcut the vector, instead only cutting the insert in three places to yieldfour small fragments from the insert. Thus, the digested vector migratedaround 2.9 kB on the agarose gel without contamination from other DNAfragments. The Hind III/Xba I/Pst I-digested vector was then ligatedwith Hind III/Xba 1-digested error-prone PCR products. After ethanolprecipitation, the ligation mixture was ready for transformation.

Transformation of Ligation Mixtures

Since the T7 promoter was used for the mutant library, an E. coli celllysogenized with lambda DE3 was utilized as the host cell for mutantenzyme expression. Specifically, a 5K(DE3) E. coli strain was used formutant library construction. First, electroporation-competent 5K(DE3)cells were made as follows: 2.5 mL of overnight cell culture was addedto 500 mL of LB broth in a 2 L sterile flask. The culture was incubatedat 37° C. on a shaker until the OD₆₀₀ reached 0.5 to 0.8. The cells werethen incubated on ice for 10 min, followed by centrifugation at 4° C.for 10 min. After washing the cells once with 500 mL ice-cold water, thecells were resuspended in 1-2 mL of 10% ice-cold glycerol. Aliquots (50μL) were made in sterile eppendorf tubes, and immediately frozen in dryice. The competent cells were stored at −80° C.

For transformation, 1 μL of ligation mixture was added to 40 μL ofcompetent cells, and the sample was transferred into an electroporationcuvette with a 0.1 cm gap. A voltage of 1.7 kv/cm was used forelectroporation. The cells were plated onto LB plates in the presence ofampicillin and incubated overnight at 37° C.

DNA Sequence Analysis of the “Xba” Mutant Library

Nine (9) mutant colonies were randomly picked for DNA sequencinganalysis. After sequencing, the number of mutations produced, thelocation of mutations, and the particular types of mutations observedwere analyzed for each mutant. The analysis revealed that all types ofbase substitutions were present in the mutants, indicating lack of biasfor a particular mutation type. In addition, only one deletion mutationwas observed in the 9 mutant clones analyzed; no base insertionmutations were identified. This indicated that the frequency of deletionand insertion in the mutant libraries was very low. The average mutationrate was 4.2 point mutations per kB. GDH activity measurements showedthat about 15-25% of the mutants in the library were active.

Example 2 Construction of the “Sma-Library” Random Mutagenesis Targetingthe α-Subunit and a Portion of the β-Subunit of GDH

A second random mutant library targeting primarily the Klebsiellapneumonia dhaB1 gene was generated. Representative sequence analysis ofthe library demonstrated that there were approximately 4.5 pointmutations per kB; enzyme activity measurements determined that about15-25% of the mutants in the library were active.

Error-Prone PCR Amplification and Mutant Library Construction

The following primers were used to amplify, by error-prone PCR usingpGD20 as template, the entire dhaB1 gene and an approximately 200 bpportion of the dhaB2 gene: DHA-F1 (SEQ ID NO:5) and DHA-R2 (SEQ ID NO:7). Error-prone PCR reactions were performed using a Clontechmutagenesis kit, as described in EXAMPLE 1. The 1.9 kB PCR products werethen digested with Hind III and Sma I.

To prepare the vector, the pGD20 plasmid was digested with Hind III andSma I (to remove the wild-type dhaB1 gene and a portion of the dhaB2gene) and then purified from an agarose gel. The Hind III/Sma I-digestederror-prone PCR product was ligated with the Hind III/Sma I-digestedpGD20 vector. The ligation mixtures were transformed into E. coli5K(DE3) by electroporation, in a manner similar to that described forcreation of the mutant library in EXAMPLE 1.

DNA Sequence Analysis of the “Sma” Mutant Library

Ten (10) mutant colonies were randomly picked for DNA sequencinganalysis, to examine the integrity of the library. For each mutant, thenumber of mutations produced, the location of mutations, and theparticular types of mutations observed were determined. Based on theseresults, the average mutation rate in the Sma-library was determined tobe 4.5 point mutations per kB. There was no apparent bias for anyparticular mutation type, and the frequency of deletion and insertion inthe mutant library was very low. Enzyme activity measurements revealedthat approximately 15-25% of the mutants in the library were active.

Example 3 Regional Random Mutagenesis Targeting Amino Acids No. 141-152(the “PpuMI-library”), 219-226 (the “4BR1-library”) and 330-342 (the“RsrII-library”) of the α-Subunit of GDH

Based on the crystal structure of GDH (Liao et al., J. InorganicBiochem. 93 (1-2): 84-91 (2003); Yamanishi et al., Eur. J. Biochem. 269:4484-4494 (2002)), the following regions of the α-subunit of GDH weretargeted for regional random mutagenesis: 1) amino acids No. 141-152; 2)amino acids No. 219-226; and 3) amino acids No. 330-342. Since each ofthese regions was fairly short in length, an oligo-directed mutagenesisapproach was used to make these three mutant libraries. This involved amulti-step process wherein a silent mutation corresponding to a uniquerestriction site upstream or downstream of each region to be mutated wasfirst created to facilitate cloning. Then, degenerate oligonucleotideprimers were prepared and used in PCR reactions to mutagenize thetargeted regions of the α-subunit. These mutagenized PCR fragments werethen cloned into E. coli to create the “PpuMI-library”, “4BR1-library”,and “RsrII-library”.

Introducing Silent Mutations in pGD20

One silent mutation was produced upstream or downstream of each targetedregion, for the creation of a unique restriction site in plasmid pGD20.

The following pairs of primers were used for making point mutations foreach region. The nucleotide shown in capitalized, boldface lettering ineach primer shows the location of the specific mutation to beintroduced. TABLE 5 Formation of Silent Mutations within Targeted AminoAcids Amino Acid RE Site and Regi n L cati n F rward Primer ReversePrimer a.a.141-a.a.152 PpuM I; 14 bp pGD20RM-F1: PGD20RM-R1: upstream of5′-gaa gat 5′-tgg ttg amino acid gcg tgc ccg gag ggg gtc target regioncAg gac ccc cTg cgg gca ctc caa cca-3′ cgc atc ttc-3′ (SEQ ID NO: 8)(SEQ ID NO: 9) a.a.219-a.a.226 HpaI; 4 bp TB4BF: TB4BR: upstream of5′-gag ctg 5′-ctc ggc amino acid ggc atg cgt gta gct ggt target regionggG tta acc taa Ccc acg agc tac gcc cat gcc cag gag-3′ ctc-3′ (SEQ IDNO: 10) (SEQ ID NO: 11) a.a.330-a.a.342 Rsr II; 11 bp pGD20RM-F2:pGD20RM-R2: downstream of 5′-act cgg 5′-tca ggg amino acid ata ttc gcctgc gcg cgg target region gGa ccg cgc tCc ggc gaa gca ccc tga-3′ tat ccgagt-3′ (SEQ ID NO: 12) (SEQ ID NO: 13)The mutagenesis experiments were carried out using Stratagene'sQuikChange site-directed mutagenesis kits (La Jolla, Calif.), accordingthe manufacturers' instructions. Following mutagenesis, plasmid waspurified from each mutant clone. The point mutations were confirmed byrestriction enzyme digestion, followed by direct DNA sequence analysis.Oligo-Directed Mutagenesis

To make the regional random mutant libraries, three degenerateoligonucleotides were synthesized (pGD20RM-F3, TB4B-R1, and pGD20RM-R4,as shown below). Normal conditions were used for oligonucleotidesynthesis for those nucleotides shown in capital letters. In contrast,nucleotides shown in lowercase, boldface text utilized the degeneratenucleotide mixtures shown beneath each primer during synthesis. Thus,1-2 point mutations were predicted to result in each “degenerate region”of the primer.

pGD20RM-F3 (for a.a.141-a.a.152 region—“PpuMI library”): (SEQ ID NO: 15)5′-GCC CGC AGG ACC CCC TCC aac cag tgc cac gtc acc aat ctc aaa gat aatccg GTG CAG ATT-3′

a=94% A mixed with 2% G, 2% C and 2% T;

g=94% G mixed with 2% A, 2% C and 2% T;

c=94% C mixed with 2% G, 2% A and 2% T;

t=94% T mixed with 2% G, 2% C and 2% A.

TB4B-R1 (for a.a.219-a.a.226 region—“4BR1 library”): (SEQ ID NO: 16)5′-GCG TGG GTT AAC cag cta cgc cga gac ggt gtc ggt cta cGG CAC CGA AGCGGT ATT TAG C-3′

a=94% A mixed with 2% G, 2% C and 2% T;

g=94% G mixed with 2% A, 2% C and 2% T;

c=94% C mixed with 2% A, 2% T and 2% G;

t=94% T mixed with 2% A, 2% G and 2% C.

pGD20RM-R4.(for a.a.330a.a.342 region—“RsrII library”): (SEQ ID NO: 17)5′-GGT GCG CGC GGT GCG GCG AAT ATC cga gtg gga gaa agt ctg gtc gtt ggcgga cgc cac ttc GAG GTC GAG-3′

a=95% A mixed with 1.66% G, 1.66% C and 1.66% T;

g=95% G mixed with 1.66% A, 1.66% C and 1.66% T;

c=95% C mixed with 1.66% G, 1.66% A and 1.66% T;

t=95% T mixed with 1.66% G, 1.66% C and 1.66% A.

High fidelity PCR reactions were then performed to produce mutagenic PCRfragments using the following primer pairs:

-   -   PpuMI-library: pGD20RM-F3 and DHA-R2 (SEQ ID NOs: 15 and 7);    -   4BR-1 library: TB4B-R1 and GD-C (SEQ ID NOs: 16 and 14);    -   RsrII-library: DHA-F1 and pGD20RM-R4 (SEQ ID NOs: 5 and 17).        The PCR fragments were purified from an agarose gel and then        digested with PpuM I/Xba I (for the PpuMI-library), HpaI/XbaI        (for the 4BR-1 library), and Hind III/Rsr II (for the        RsrII-library), respectively.        Mutant Library Construction

The mutated pGD20 construct (in which PpuM I, HpaI or Rsr II uniquerestriction enzyme digestion sites had been introduced by site-directedmutagenesis), was digested with PpuM I/Xba I, HpaI/XbaI or Hind III/RsrII, respectively. The linearized vectors were purified from agarosegels, and then ligated with the restriction enzyme-digested PCRproducts. Mutant libraries were prepared by electroporating the ligationmixtures into E. coli strain 5K(DE3), as described in EXAMPLE 1.

Sequencing Analysis for Regional Libraries

Between 9 and 10 mutant colonies were randomly picked for DNA sequencinganalysis from each regional library. For the PpuMI-library, the averagemutation rate per mutant is 2.8 mutations (n=9). The average mutationrate per mutant is 2.0 mutations in the 4BR1-library (n=8). Finally,sequencing data from the Rsr II library revealed an average mutationrate of 2.2 mutations per mutant (n=10). Neither insertions nordeletions were observed in any of the three libraries. All types of basesubstitutions were detected, however, indicating lack of bias for anyspecific mutation type.

Example 4 Screening Assay Development

A screening assay was developed, wherein a B₁₂-dependent dehydratasereaction could be manually “started” by the addition of B₁₂ coenzyme andsubstrate (glycerol). The dehydratase reaction product, 3-HP, isdetected by a colorimetric aldehyde assay. Using this assay, preliminaryanalyses concerning the time course of typical B₁₂-dependent dehydratasereactions in the presence of glycerol and 1,3-propanediol alloweddevelopment of a rapid theoretical technique to estimate the initialrate of the reaction, v, and the observed enzyme inactivation rate,k_(inact obsd).

Screening Assay Rationale

Cultures expressing either wild-type B₁₂-dependent dehydratase or mutantB₁₂-dependent dehydratase produce apo-B₁₂-dependent dehydratase if theyhave no source of the enzyme's cofactor, coenzyme B₁₂. In contrast,holoenzyme forms spontaneously if coenzyme is added to toluene- anddetergent-permeabilized whole cells. It is therefore possible to “start”a B₁₂-dependent dehydratase reaction by adding coenzyme, plus substrate,to such cells. The reaction product, 3-HP, can be detected by acalorimetric aldehyde assay (Zurek, G., Karst, U. Analytica ChimicaActa, 351:247-257 (1997)) using the reagent 3-methyl-2-benzothiazolinone(MBTH) and an oxidant, ferric chloride.

Preliminary Assays to Examine the Time Course of Typical GDH Reactions(with and without 1,3-propanediol)

Cells producing wild-type GDH were grown overnight in 15% Lennox broth(made by diluting 15 volumes of Lennox broth (Gibco-BRL, Rockville, Md.)with 85 volumes of 0.5% NaCl, followed by sterilization) at 37° C. withshaking (250 rpm) in an Innova 4300 incubator (New Brunswick Scientific,New Brunswick, N.J.). Cells were permeabilized as follows: aliquots ofculture (0.3 mL) were dispensed into the square wells of a polypropylenedeep-well plate (Beckman-Coulter, Fullerton, Calif.) and 10 μL oftoluene containing 2.5% (v/v) Triton X-100 detergent was added to eachwell. Plates were then shaken for 10 min at top speed on an IKA MTS4shaker (IKA-Werke Gmbh., Staufen, Germany). Working under red light toprotect the coenzyme B₁₂, reactions were started by adding 5 volumes ofsubstrate solution to 1 volume of permeabilized cell suspension. Thesubstrate solution contained 12 mM glycerol and 24 μM coenzyme B₁₂ in0.1 M K-Hepes, pH 8. At timed intervals, 12.5 μl aliquots of thereaction were added to the wells of a 96-well plate which contained 12.5μl of 3 mg/mL MBTH in 0.4 M glycine-HCl, pH 2.7. At least 20 min aftersampling, 125 μL of 5.5 mM FeCl₃ in 10 mM HCl was added to each of theMBTH-containing samples. After a further 20 min or longer, the bluecolor associated with GDH activity was quantitated as absorbance at 670nm using a Spectramax 160 plate reader (Molecular Devices, Sunnyvale,Calif.). A similar experiment was conducted in which the substratesolution was supplemented with 50 mM 1,3-propanediol.

Theoretical Analysis of Assay Results

Data from the time course reactions were plotted as time versus OD₆₇₀,as shown in FIG. 1. Specifically, the upper trace in FIG. 1 shows thetime course of the GDH reaction (room temperature, in 0.1 M K-HEPES, pH8) with 10 mM glycerol (K_(m) ˜0.5 mM) as substrate. The rate of productformation decreases rapidly with time as inactivation occurs. Atheoretical curve was drawn through the points by fitting threeparameters to the following equationy=T₀+amp(1−exp(−k_(inact obsd)*time)). These parameters were: 1.) abackground (t=0 (T₀)) value for the assay; 2.) an amplitude value (amp)for the limiting total absorbance produced during the assay; and 3.) afirst-order inactivation rate constant (k_(inact obsd)), which controlsthe curvature. The fitting parameters are related to the kineticproperties of the reaction as follows: the amplitude is equal to theinitial rate of the reaction (v) divided by the observed enzymeinactivation rate (k_(inact obsd)). The initial rate is[GDH][gly]k_(cat)/(K_(m)+[gly]). If the assay background is subtracted,further analysis reveals that:

-   -   1. Both v and k_(inact obsd) can be estimated from only two        samples taken during the reaction: an early point (T1) for        estimation of v, and a late point (T2) for the amplitude; and    -   2. 1/k_(inact obsd) can be estimated as T2/T1.

The lower trace of FIG. 1 shows the effect of including 50 mM1,3-propanediol (K_(i)˜15 mM) in the assay. The initial rate of thereaction is reduced only ˜20%, but inactivation was ˜3 times faster.This is because, although the competitive inhibition is modest, the rateconstant for inactivation of enzyme-1,3-propanediol complexes(k_(inact 1,3-propanediol)) is greater than that for enzyme-glycerolcomplexes (k_(inact glycerol)).

A kinetic scheme, which incorporates both inactivation processes, isshown below.

Example 5 Two-Point High Throughput Screening Assay

Mutant colonies prepared in EXAMPLES 1, 2, and 3 were examined using ahigh throughput screening assay protocol that was based on themethodology described in EXAMPLE 4. This assay specifically measured theGDH reaction product at two time-points during the assay: T1, measuredat 30 sec after T₀; and T2, measured at 40 min after T₀.

Colonies from plated libraries were picked into 94 wells in a standard96-well micro-titer plate containing 0.15 mL/well of 15% Lennox broth.The remaining two wells were inoculated with cells producing wild-typeGDH, and the cells were allowed to grow at 37° C. in a static incubatorfor 4-6 hr. Alternatively, if storage of the picked cells at −80° C.before screening was desired, the medium also included glycerol (10%v/v), and the picked cells were allowed to grow overnight before beingstored. Before screening, previously frozen cells were transferred witha 96-pin inoculator (V&P Scientific, San Diego, Calif.) into fresh96-well plates containing 15% Lennox broth without glycerol and allowedto grow for 6-16 hr at 37° C. without shaking.

For GDH mutant screening, the cell growth protocol was as follows: 15%Lennox broth medium was dispensed (0.3 mL/well) into the wells ofpolypropylene deep-well 96-well plates (Beckman-Coulter, Fullerton,Calif.). Cells were inoculated from the shallow 96-well plates into thedeep wells using a 96-pin long-pin inoculator (V&P Scientific), and theplate was covered with 3″ wide Micropore surgical tape (3M Health Care,St. Paul, Minn.). The shallow 96-well plates were stored at 4° C. untilthe assays' completion, when they were used as a source of viable cellsfor further examination of lines identified as potentially improved. Thecells in the deep-well plates were allowed to grow with shaking (250rpm) at 37° C. overnight. The air in the incubator (Innova 4300, NewBrunswick Scientific, New Brunswick, N.J.) was humidified with a wetsponge in a plastic tray.

Cells were permeabilized in the 96-well plates by adding 10 μL oftoluene containing 2.5% (v/v) Triton X-100 detergent to each well.Plates were then shaken for 10 min at top speed on an IKA MTS4 shaker(IKA-Werke Gmbh., Staufen, Germany). Aliquots (8 μL) of permeabilizedcells were transferred into 96-well reaction plates using a Biomek 2000robot (Beckman-Coulter), located in a Plexiglas® (Rohm and Haas,Philadelphia, Pa.) enclosure covered in red plastic film to protect thesubstrate mixture from white room light. Reaction at room temperaturewas initiated by robotic addition to the cells of 40 μL of substratemixture (prepared under red light, and stored at −20° C. in foil-wrappedcontainers until needed) containing 24 μM coenzyme B₁₂, 12 mM glycerol,and 50 mM 1,3-propanediol in 0.1 M potassium-HEPES buffer, pH 8. Thirtyseconds after substrate addition, a 12.5 μL aliquot of the reaction (theT1 sample) was transferred into a second plate whose wells contained12.5 μL of 3-methyl-2-benzothiazolinone (MBTH) in 0.4 M glycine-HCl, pH2.7. Approximately 40 minutes after substrate addition, a similaraliquot of the reaction (the T2 sample) was transferred to a third platecontaining MBTH. At least 20 min after this transfer, 125 μL of asolution of 5.5 mM FeCl₃ in 10 mM HCl was added to each well of thesecond and third (MBTH-containing) plates. After a further 20-60 min,the blue color associated with GDH activity was quantitated asabsorbance at 670 nm using a Spectramax 160 plate reader (MolecularDevices, Sunnyvale, Calif.).

Data from the plate reader were transferred to a modified Microsoft®(Redmond, Wash.) Excel computer program. The program was formatted tomatch the first (T1) and second (T2) samples from each reaction plateand to prepare output as tables of results showing plate, locationwithin plate, and T1, T2, and T2/T1 ratio for each reaction. The datawere examined for samples showing exceptionally high values for T1, T2,or T2/T1 ratio. This was facilitated by the ability of the program tosort the data by any of these parameters. The most promising candidateswere streaked out from the retained shallow 96-well plates for furtherexamination.

Example 6 Screening the GDH Mutant Libraries and Identifying PositiveHits

Using the automated high throughput assay described in EXAMPLE 5,approximately 100,000 mutant colonies from 5 mutant libraries werescreened. These libraries included:

-   -   1.) the Xba library, targeting the α-, β-, and γ-subunits        (DhaB1, DhaB2, and DhaB3), as described in EXAMPLE 1;    -   2.) the Sma library, targeting the α- and a small portion of the        β-subunits, as described in EXAMPLE 2;    -   3.) the PpuMI library, targeting a.a.141-a.a.152 of the        α-subunit, as described in EXAMPLE 3;    -   4.) the 4BR1 library, targeting a.a.219-a.a.226 of the        α-subunit, as described in EXAMPLE 3; and    -   5.) the RsrII library, targeting a.a.330a.a.342 of the        α-subunit, as described in EXAMPLE 3.        All individual isolates were derived from 1 of the 5 libraries        described above. Most individual isolates were uniquely        identified by a label indicating the source library, followed by        a number (e.g. “Xba3010”). All putative “hits” from the first        screen were confirmed in follow-up assays. Most mutational        effects were classified according to 4 broad categories based on        kinetic parameters as described below. Sequence analysis of the        mutant genes, followed by comparison with the wild-type gene,        permitted identification of the specific point mutations present        in each mutant gene.        Screening the Mutant Libraries and Confirming the Hits

Following the primary screening of approximately 100,000 mutants,putative hits were confirmed by a follow-up confirmation assay. Briefly,each putative hit was re-assayed in 8 wells. Results from eachindividual clone were analyzed statistically to obtain the mean andstandard deviation for T1 (the amount of aldehyde measured at 30 sec)and T2 (the amount of aldehyde measured at 40 min). These results werecompared to the wild-type enzyme.

FIG. 2 shows a typical follow-up assay result, plotting the number ofassays on the x-axis versus the OD_(670nm) on the y-axis. Results fromeach individual assay (n=8) at T1 and T2 for clone Xba3010 and thewild-type are presented. The mean value is graphically shown as ahorizontal line, and numerically presented in parentheses +/−standarddeviation. Generally, the standard deviation was about 10-15%.

Screening Results

TABLE 6 summarizes the follow-up assay results for hits having either aT2/T1 ratio greater than that of the wild-type, or a T2 greater thanthat of the wild-type, or both. The T2/T1 ratio provides an indicationof enzyme stability during the 40 min reaction, which occurs in thepresence of 1,3-propanediol and glycerol. T2 indicates the totalturnover number of the enzyme before it becomes completely inactivated.The higher the T2/T1 ratio, the better the enzyme's stability. Thus, amutant enzyme with improved stability will have a decreased rate ofinactivation in the presence of glycerol and 1,3-propanediol, ascompared to the wild-type enzyme.

In TABLE 6, the mean values of T1 and T2 from the follow-up assays arereported, following their normalization to the wild-type results. Mostmutant enzymes are categorized as Type-1, -2, -3, or 4 mutants, based onthe following definitions:

-   -   Type 1 mutants: T2/T1 ratio is improved with respect to        wild-type, while the absolute T2 value is decreased;    -   Type-2 mutants: both T2/T1 ratio and T2 are improved over the        wild-type, but the degree of T2 improvement is less than that of        T2/T1 ratio improvement;    -   Type-3 mutants: both T2/T1 ratio and T2 are improved over the        wild-type, and the degree of improvement in both are similar;        and    -   Type-4 mutants: T2/T1 ratio is equivalent or reduced with        respect to wild-type, but T2 is improved.

TABLE 6 also summarizes the specific point mutations identified in eachmutant. The SEQ ID NO: of the DNA sequence of the enzyme is provided inthe first column of the Table. TABLE 6 Summary of Type-1, Type-2,Type-3, and Type-4 Mutants T2/T1 T2 Strain Ratio* value* Mutation(s)Wild-type (control) 1 1 none (SEQ ID NO: 1) Type 1 Xba3007 4.30 0.78ACC(γ-Thr53) to GCC(Ala) (SEQ ID NO: 40) Xba3029 3.24 0.91 CTC(α-Leu509)to TTC(Phe) (SEQ ID NO: 44) Xba3004 2.88 0.63 ATG(α-Met306) to CTG(Leu);(SEQ ID NO: 48) ACT(β-Thr45) to GCT(Ala); AAC(β-Asn155) to AGC(Ser)Xba3025 2.77 0.84 ATC(γIle49) to ACC(Thr) (SEQ ID NO: 52) Xba3038 2.240.58 TTT(α-Phe233) to CTT(Leu) (SEQ ID NO: 56) Xba3030 2.00 0.59ATG(αMet257) to GTG(Val) (SEQ ID NO: 60) Xba3006 1.97 0.51 GTG(α-Val44)to GCG(Ala); (SEQ ID NO: 64) ACC(α-Thr470) to GCC(Ala) Xba3031 1.92 0.64GTC(α-Val226) to GCC(Ala) (SEQ ID NO: 68) Xba3017 1.90 0.69ATC(α-Ile105) to ATT(Ile); (SEQ ID NO: 72) TCA(α-Ser168) to CCA(Pro)Xba3005 1.84 0.72 ATC(α-Ile67) to GTC(Val); (SEQ ID NO: 76)GAG(α-Glu209) to GAA(Glu); AAC(β-Asn155) to AAG(Lys) Xba3033 1.75 0.72ATG(α-Met257) to ACG(Thr); (SEQ ID NO: 80) GAC(β-Asp181) to GGC(Gly)Xba3032 1.60 0.91 TAC(α-Tyr70) to AAC(Asn); (SEQ ID NO: 84) GTG(α-Val86)to GAG(Glu) Xba3018 1.60 0.79 TAC(α-Tyr70) to AAC(Asn); (SEQ ID NO: 88)GTT(α-Val74) to GTC(Val) Xba3014 1.57 0.66 CCG(α-Pro430) to TCG(Ser);(SEQ ID NO: 92) GAA(β-Glu25) to GAG(Glu); AAA(γ-Lys27) to AGA(Arg)Xba3024 1.44 0.84 GTG(α-Val44) to GAG(Glu); (SEQ ID NO: 96)GTG(α-Val461) to GGG(Gly) Xba3026 1.43 0.93 ACC(α-Thr350) to GCC(Ala)(SEQ ID NO: 100 Sma3009 2.75 0.71 ATG(α-Met62) to GTG(Val); (SEQ ID NO:104) ATC(α-Ile63) to GTC(Val); AAA(α-Lys149) to AGA(Arg) Sma3010 2.030.96 ATG(α-Met62) to GTG(Val); (SEQ ID NO: 108 GCG(β-Ala53) to GTG(Val)Sma3014 1.84 0.73 CAG(α-Gln59) to CGG(Arg); (SEQ ID NO: 112)ATT(α-Ile314) to GTT(Val); TTT(β-Phe11) to TTA(Leu) Sma3008 1.75 0.95ATG(α-Met62) to ACG(Thr); (SEQ ID NO: 116 CTG(α-Leu268) to CTA(Leu)Sma3001 1.56 0.97 AAC(α-Asn520) to AGC(Ser) (SEQ ID NO: 120 PpuMI0012.43 0.57 CGG(α-Arg137) to AGG(Arg); (SEQ ID NO: 124) TGC(α-Cys143) toTGT(Cys); CTC(α-Leu148) to CGC(Arg); CCG(α-Pro152) to CCC(Pro) PpuMI0022.08 0.69 CGG(α-Arg137) to AGG(Arg); (SEQ ID NO: 128 GAT(α-Asp150) toCAT(His) PpuMI005 1.85 0.81 CGG(α-Arg137) to AGG(Arg); (SEQ ID NO: 132)CAG(α-Gln242) to CAA(Gln); AAA(α-Lys149) to CAA(Gln); CCG(α-Pro152) toCCC(Pro) RsrII001 1.92 0.72 TTC(α-Phe339) to GTC(Val); (SEQ ID NO: 136CGC(α-Arg346) to CGG(Arg) Type 2 Sma3002 2.73 1.60 TAT(α-Tyr271) toTGT(Cys); (SEQ ID NO: 140) TAC(α-Tyr502) to CAC(His); TAA(stop of α) toCAA(Gln); CAA(β-Gln2) to CGA(Arg); TTT(β-Phe11) to TTC(Phe) Sma3003 1.951.16 ATG(α-Met62) to CTG(Leu) (SEQ ID NO: 143) Xba3015 2.36 1.13GTT(α-Val549) to GCT(Ala); (SEQ ID NO: 147) CTG(β-Leu113) to CCG(Pro);GCC(γ-Ala122) to GTC(Val); GCG(γ-Ala128) to GTG(Val) Xba3008 2.12 1.15TCT(β-Ser122) to CCC(Pro); (SEQ ID NO: 151 AAA(β-Lys166) to AGA(Arg)Xba3016 1.72 1.18 ATC(α-Ile102) to ACC(Thr) (SEQ ID NO: 155 Xba3020 1.651.04 CCG(β-Pro152) to ACG(Thr) (SEQ ID NO: 159 Xba3037 1.48 1.06GAG(α-Gly116) to GAA(Glu); (SEQ ID NO: 163 GTT(α-Val423) to ATT(Ile)Xba3036 1.27 1.03 GGT(α-Gly47) to GGC(Gly); (SEQ ID NO: 167 CGA(α-Arg65)to CAA(Gln) 4BR1001 1.90 1.10 GGC(α-Gly216) to GGG(Gly); (SEQ ID NO: 171GTG(α-Val224) to CTG(Leu) Type 3 Xba3010 1.25 1.39 CTG(α-Leu318) toTTG(Leu); (SEQ ID NO: 175) AAC(α-Asn447) to AAT(Asn); AAT(α-Asn489) toAGT(Ser); GCC(β-Ala27) to TCC(Ser) Type 4 Xba3009 0.98 1.61 ACG(α-Thr77)to GCG(Ala); (SEQ ID NO: 179) TGC(α-Cys193) to AGC(Ser); TAA(stop of α)to GAA(Glu); AAA(β-Lys56) to AGA(Arg); GCC(β-Ala88) to GCT(Ala);GAT(β-Asp111) to GAA(Glu); CAT(γ-His67) to TAT(Tyr); ACC(γ-Thr114) toTCC(Ser) Xba3023 1.00 1.22 GGG(α-Gly63) to GGA(Gly); (SEQ ID NO: 182)CAC(α-His96) to CAT(His); ATC(α-Ile102) to GTC(Val) Other KG002 1.031.22 CTG(β-Leu113) to CCG(Pro); (SEQ ID NO: 241) ACC(γ-Thr114) toGCC(Ala); GCT(α-Ala309) to GCC(Ala); AAC(α-Asn468) to AAT(Asn) KG0031.05 0.92 GTG(α-Val224) to ATG(Met); (SEQ ID NO: 245) CCG(α-Pro450) toCCA(Pro) KG004 1.53 1.14 GTC(α-Val226) to GCC(Ala); (SEQ ID NO: 249)ATG(α-Met306) to TTG(Leu) KG005 1.62 1.33 ACT(α-Asn288) to ACC(Asn);(SEQ ID NO: 253) ATG(α-Met306) to TTG(Leu); CCG(β-Pro152) to TCG(Ser)KG006 1.09 1.22 ATG(α-Met62) to ACG(Thr) (SEQ ID NO: 257) KG007 1.111.16 GTC(α-Val115) to GCC(Ala); (SEQ ID NO: 261) CTG(β-Leu13) toCCG(Pro) KG010 1.15 1.06 AAT(α-Asn151) to AAC(Asn); (SEQ ID NO: 265)TTC(α-Phe513) to CTC(Leu) KG011 1.35 1.03 ATG(α-Met214) to TTG(Leu);(SEQ ID NO: 269) GCG(α-Ala460) to GCA(Ala); GAA(α-Glu462) to GAG(Glu);ACC(α-Thr499) to GCC(Ala); GAT(β-Asp24 to GGT(Gly); GAA(β-Glu29) toGAG(Glu); CTG(β-Leu58) to CTT(Leu); CGG(β-Arg70) to CGA(Arg);GAG(β-Glu130) to GGG(Gly); AAA(γ-Lys4) to AAG(Lys) KG012 1.15 1.01TTA(α-Leu217) to GTA(Val) (SEQ ID NO: 273) KG014 1.1 0.95 ATG(α-Met62)to GTGVal); (SEQ ID NO: 277) GAT(β-Asp24) to GAA(Glu) KG016 0.97 1.02CGG(α-Arg137) to AGG(Arg); (SEQ ID NO: 281) AAC(α-Asn141) to ATC(Ile);GAT(α-Asp150) to GAC(Asp); GCG(α-Ala231) to ACG(Thr); GGC(α-Gly236) toAGC(Ser) KG017 0.93 1.05 TCA(α-Ser41) to TCG(Ser); (SEQ ID NO: 285)GCG(α-Ala119) to ACG(Thr); AAC(α-Asn447) to AAT(Asn) KG021 1.29 0.93GTC(α-Val226) to GCC(Ala) (SEQ ID NO: 289) KG023 1.21 0.88 CCG(β-Pro152)to TCG(Ser) (SEQ ID NO: 293) KG001 5.7 0.80 AGC(α-Ser219) to AAC(Asn)(SEQ ID NO: 297)*The T2/T1 ratio and T2 value are relative numbers normalized to thewild-type.

As seen from the sequencing results, most mutations were identified asamino acid substitutions (with the exception of the silent mutations).However, two of the mutants (Sma3002 [SEQ ID NO:140] and Xba3009 [SEQ IDNO:179]) were fusion proteins. Specifically, the stop codon (TAA) of thegene encoding the α-subunit (dhaB1) was changed to CAA (Gln) or GAA(Glu) in Sma3002 and Xba3009, respectively. Since there are 15 bpbetween this stop codon and the initial codon of the gene encoding theβ-subunit (dhaB2), neither of these fusion proteins caused frame shiftsto occur in the β-subunit. This permitted both mutant enzymes to retainactivity. The initial codon (GTG) of the β-subunit is usually recognizedby fMet-tRNA; however, in the fusion mutants, this codon should berecognized by Val-tRNA. The Sma3002 fusion protein contained a linkerthat consists of six amino acid residues: Gln-Gly-Gly-Ile-Pro-Val (SEQID NO:18). For the Xba3009 fusion protein, the linker wasGlu-Gly-Gly-Ile-Pro-Val (SEQ ID NO:19).

Example 7 Biochemical Analysis of the Mutants Using Purified Enzyme

Better characterization of five mutants and the wild-type GDH wasaccomplished following each enzyme's over-expression and purification.

Over-Expression and Purification

Xba3007 (Type-1; SEQ ID NO:40), Sma3002 (Type-2; SEQ ID NO:140), Xba3010(Type-3; SEQ ID NO:175), Xba3009 (Type-4; SEQ ID NO:179) and 4BR1001(Type-2; SEQ ID NO:171)) were selected for further biochemical analysis,along with the wild-type enzyme. First, plasmids were purified from the5K(DE3) E. coli host strain, and transformed into E. coli BL21 (DE3).The cells were grown in LB medium containing ampicillin to an OD₆₀₀ of0.6-1.0 before 1.0 mM IPTG was added. After 3 hr of induction at 37° C.,cells were harvested by centrifugation and washed once with 20 mMHEPES-KOH buffer (pH 8.0). The cell pellets were stored at −80° C.

For enzyme purification, the cell pellets were first resuspended in 20mM HEPES-KOH buffer (pH 8.0) containing a Complete Mini proteaseinhibitor cocktail tablet (Roche, Polo Alto, Calif.) and 0.5 mM EDTA.The cells were broken by sonication (Branson model 450; 20% output, 50%pulse, 4 min in ice bath), followed by centrifugation (40,000×g, 30 min,4° C.). The clear supernatants were spun again (110,000×g, 1 hr, 4° C.),and (NH₄)₂SO₄ was slowly added into the supernatant on ice to bring thesolution to 50% saturation. The solutions were stirred for 25 min onice, followed by centrifugation (40,000×g, 30 min, 4° C.). The pelletswere resuspended in 2 mL of running buffer (100 mM HEPES-KOH (pH 8.2),100 mM 1,2-propanediol and 1 mM DTT), and then applied to a 16/60 HiloadSuperdex200 size exclusion column (Pharmacia Biotech, Piscataway, N.J.)equilibrated with the running buffer.

The enzymes were eluted with running buffer at a flow rate of 0.25mL/min, and the eluents were collected using a fraction collector (3mL/fraction). Fractions were assayed for enzyme activity using the assaydescribed in EXAMPLE 5, and then the active fractions were pooled andconcentrated using Centricon YM100 (Milipore, Bedford, Mass.). Theconcentrated enzymes were passed over the same column an additional timeusing fresh running buffer consisting of 100 mM HEPES-KOH (pH 8.2) and 1mM DTT. The purified enzymes were 75-95% pure, as judged by SDS-PAGEelectrophoresis using a 10-20% gradient gel and Coomassie blue staining.

Biochemical Characterization of the Mutant GDH Enzymes:

Detailed enzyme kinetic analyses using the purified wild-type and mutantGDH enzymes were conducted. The enzyme activity was determined bymeasuring product formation using the MBTH-colorimetric aldehyde assay(Zurek, G., Karst, U. Analytica Chimica Acta, 351:247-257 (1997)), andthe K_(M) and V_(max) were determined from Lineweaver-Burke plots. Thek_(cat) was calculated from V_(max). The determined K_(M) and k_(cat) ofthe enzymes are shown in TABLE 7. TABLE 7 K_(M) and k_(cat) of wild-typeand Selected Mutant GDH enzymes Enzyme K_(M) (mM) k_(cat) (min⁻¹) WT2.56 29,000 Sma3002 (Type 2) 2.94 9,830 4BR1001 (Type 2) 2.00 15,729Xba3007 (Type 1) 20.00 16,220 Xba3009 (Type 4) 3.13 65,373 Xba3010 (Type3) 3.23 28,017

Finally, a detailed analysis of each mutant enzyme's inactivationproperties was performed. The glycerol inactivation rate constant wasmeasured as described in EXAMPLE 4. To measure the air inactivation rateconstant, the enzyme was diluted to 27 μg/mL in 0.1 M K-HEPES buffer (pH8) containing 21 μM coenzyme B₁₂, and then incubated in air at roomtemperature. The total turnover number was measured after 0.25, 0.5, 1,2, 5, 10, 15, 20 and 30 min incubation. To measure the total turnovernumber, 10 μL of the enzyme solution was added to 190 μL reactionsolution containing 200 mM glycerol, 24 mM coenzyme B₁₂ and 0.1 MK-HEPES buffer (pH 8), and the reaction mixture was incubated at roomtemperature for 2 hr. The total turnover number was estimated bymeasuring 3-HP concentration using the MBTH-colorimetric aldehyde assay(Zurek, G. and Karst, U., supra). The data was plotted as time versustotal turnover number, and the air inactivation rate constant wasestimated by curve-fitting using the equation A=A₀ exp(−k_(air)t),where: “A” is the total turnover number, “A₀” is the total turnovernumber at time zero, “k_(air)” is the air inactivation rate constant,and “t” is time.

To measure 1,3-propanediol inactivation, the enzyme was diluted to 27μg/mL in 0.1 M K-HEPES buffer (pH 8) containing 21 μM coenzyme B₁₂ andvarious concentrations of 1,3-propanediol (i.e., 1, 20, 100 and 300 mM).For each 1,3-propanediol concentration, the inactivation rate constantwas estimated by the method used for measuring the air inactivation rateconstant. The inactivation rate constants were plotted against1,3-propanediol concentration, and the maximum inactivation rateconstant by 1,3-propanediol was estimated from the curve. Thedissociation constant for 1,3-propanediol was the 1,3-propanediolconcentration at which the inactivation rate constant was half of themaximum inactivation rate constant.

Results from this analysis of inactivation properties are shown below inTABLE 8. In the absence of either glycerol or 1,3-propanediol or light,but in the presence of air (oxygen), inactivation of B₁₂-dependentdehydratase holoenzymes occur with bi-phasic kinetics. k_(gly) is theinactivation rate constant measured in the presence of 10 mM glycerolwithout 1,3-propanediol. All of the mutant and wild-type dehydratasesshowed a biphasic air inactivation; thus, k_(air, F) is the airinactivation rate constant for the fast phase and k_(air, S) is the airinactivation rate constant for the slow phase. k_(1,3-propanediol) isthe maximum inactivation rate constant by 1,3-propanediol. K_(d) is thedissociation constant for 1,3-propanediol. TABLE 8 Inactivationproperties of Selected Mutant enzymes k_(1,3-) k_(gly) k_(air,F)k_(air,S) propanediol K_(d) Strain (min⁻¹) (min⁻¹) (min⁻¹) (min⁻¹) (mM)WT GDH 0.43 0.35 0.03 1.99 41 Sma3002 0.13 0.04 0.02 0.76 39 4BR1001 —0.10 0.03 1.02 16 Xba3007 0.03 0.05 0.01 0.41 40 Xba3009 0.33 0.11 0.032.76 20 Xba3010 0.45 — — — —

Example 8 Second Round Mutagenesis by Combination of Selected FirstGeneration Mutants

To further improve the mutants obtained from the first round ofmutagenesis, a second round of mutagenesis was performed to combinemutations from several first generation mutants.

To make the second generation mutants, plasmids from several firstgeneration mutants containing multiple mutations (e.g., Sma3002 orXba3009) were first purified from the host cells. The single pointmutation found in Xba3007, Xba3029 or 4BR1001 was then introduced intothese plasmids, to produce second generation mutants 2-F4,12-B1, 13-B7,and 16-H5. TABLE 9 summarizes the details concerning each of thesemutants and the primers used to produce them. The nucleotide shown incapitalized, boldface lettering in each primer shows the location of thespecific mutation to be introduced. TABLE 9 Synthesis of SecondGeneration Mutants 2^(nd) Generation “Parent” Forward Reverse MutantMutants Primer Primer 2-F4 Sma3002 + 2-F4-F1: 2-F4-R1: 4BR1001 5′-c tacgcc gag acg Ctg 5′-gcc gta gac cga caG tcg gtc tac ggc-3′ cgt ctc ggcgta g-3′ (SEQ ID NO: 20) (SEQ ID NO: 21) 12-B1 Xba3009 + 12-B1-F1:12-B1-R1: Xba3007 5-gga tct ccc gcc agG 5′-ggt act caa ggg Cct ccc ttgagt acc-3′ ggc ggg aga tcc-3′ (SEQ ID NO: 22) (SEQ ID NO: 23) 13-B7Sma3002 + 12-B1-F1: 12-B1-R1: Xba3007 5′-gga tct ccc gcc agG 5′-ggt actcaa ggg Cct ccc ttg agt acc-3′ ggc ggg aga tcc-3′ (SEQ ID NO: 22) (SEQID NO: 23) 16-H5 Sma3002 + 16-H5-F1: 16-H5-R1: Xba3029 5′-cag acc tcggcc att Ttc 5′-ctc gaa ctg ccg atc gaA gat cgg cag ttc gag-3′ aat ggccga ggt ctg-3′ (SEQ ID NO: 24 (SEQ ID NO: 25)Mutagenesis experiments were performed using Stratagene QuikChangesite-directed mutagenesis kits (Stratagene, La Jolla, Calif.), asdescribed in EXAMPLE 3.

The T2/T1 ratio and T2 value for each of the second generation mutantswas determined, as previously described in EXAMPLE 5. These results areshown below in TABLE 10. The SEQ ID NO: of the DNA sequence of theenzyme is provided in the first column of the Table. TABLE 10 T2/T1Ratio and T2 Value of Second Generation mutants Mutant Mutation T2/T1ratio* T2* WT — 1 1 (SEQ ID NO: 1) Sma3002 TAT(α-Tyr271) to TGT(Cys);2.7 1.6 (SEQ ID NO: 140) TAC(α-Tyr502) to CAC(His); TAA(stop of α) toCAA(Gln); CAA(β-Gln2) to CGA(Arg); TTT(β-Phe11) to TTC(Phe) Xba3007ACC(γ-Thr53) to GCC(Ala) 4.3 0.8 (SEQ ID NO: 40) Xba3009 ACG(α-Thr77) toGCG(Ala); 1.0 1.6 (SEQ ID NO: 179) TGC(α-Cys193) to AGC(Ser); TAA(stopof α) to GAA(Glu); AAA(β-Lys56) to AGA(Arg); GCC(β-Ala88) to GCT(Ala);GAT(β-Asp111) to GAA(Glu); CAT(γ-His67) to TAT(Tyr); ACC(γ-Thr114) toTCC(Ser) Xba3029 CTC(α-Leu509) to TTC(Phe) 3.4 0.9 (SEQ ID NO: 44)4BR1001 GGC(α-Gly216) to GGG(Gly); 1.9 1.1 (SEQ ID NO: 171)GTG(α-Val224) to CTG(Leu) 2-F4 GTG(α-Val224) to CTG(Leu); 14.4 0.5 (SEQID NO: 186) TAT(α-Tyr271) to TGT(Cys); TAC(α-Tyr502) to CAC(His);TAA(stop of α) to CAA(Gln); CAA(β-Gln2) to CGA(Arg); TTT(β-Phe11) toTTC(Phe) 12-B1 ACG(α-Thr77) to GCG(Ala); 4.6 1.8 (SEQ ID NO: 189)TGC(α-Cys193) to AGC(Ser); TAA(stop of α) to GAA(Glu); AAA(β-Lys56) toAGA(Arg); GCC(β-Ala88) to GCT(Ala); GAT(β-Asp111) to GAA(Glu);ACC(γ-Thr53) to GCC(Ala); CAT(γ-His67) to TAT(Tyr); ACC(γ-Thr114) toTCC(Ser) 13-B7 TAT(α-Tyr271) to TGT(Cys); 2.4 0.8 (SEQ ID NO: 192)TAC(α-Tyr502) to CAC(His); TAA(stop of α) to CAA(Gln); CAA(β-Gln2) toCGA(Arg); TTT(β-Phe10) to TTC(Phe); ACC(γ-Thr53) to GCC(Ala) 16-H5TAT(α-Tyr271) to TGT(Cys); 16.8 1.2 (SEQ ID NO: 195) TAC(α-Tyr502) toCAC(His); CTC(α-Leu509) to TTC(Phe); TAA(stop of α) to CAA(Gln);CAA(β-Gln2) to CGA(Arg); TTT(β-Phe11) to TTC(Phe)*The T2/T1 ratio and T2 value are relative numbers, normalized to thewild-type enzyme.Those mutants shown in bold text are second generation mutants.

Example 9 Construction and Analysis of the Pure Fusion Mutants

Two fusion mutants, Xba3009 (SEQ ID NO:179) and Sma3002 (SEQ ID NO:140),were described in EXAMPLE 6. Both contained other mutations, in additionto the fusion itself. In order to investigate the effects of the α- andβ-fusion, two pure fusion mutants (1E1 and 20G7) were constructed.

Construction of Pure Fusion Mutants

The stop codon of the α-subunit (TAA) was changed to CAA (1E1) or GAA(22-G7). This modification was achieved by introducing the single pointmutation into the wild-type GDH plasmid. The following two pairs ofprimers were used for making the point mutations: For the 1-E1 mutant:1-E1-F1: (SEQ ID NO:26) 5′-gac acc att gaa Caa ggc ggt att cct-3′1-E1-R1: (SEQ ID NO:27) 5′-agg aat acc gcc ttG ttc aat ggt gtc-3′ Forthe 22-G7 mutant: 22-G7-F1: (SEQ ID NO:28) 5′-ccc gac acc att gaa Gaaggc ggt att cct gtg-3′ 22-G7-R1: (SEQ ID NO:29) 5′-cac agg aat acc gccttC ttc aat ggt gtc ggg-3′The nucleotide shown in capitalized, boldface lettering in each primershows the location of the specific mutation to be introduced.

Mutagenesis experiments were carried out using a Stratagene QuikChangesite-directed mutagenesis kit (Stratagene, La Jolla, Calif.), asdescribed in EXAMPLE 3. The T2/T1 ratio and T2 value of these twomutants were measured as described in EXAMPLE 5. These results are shownin TABLE 11. The SEQ ID NO: of the DNA sequence of the enzyme isprovided in the first column of the Table. TABLE 11 T2/T1 Ratio and T2Value of Pure Fusion Mutants T2/T1 Mutant Mutation ratio* T2* WT — 1 11-E1 TAA (stop of α) to 0.72 1.82 (SEQ ID NO:198) CAA (Gln) 22-G7 TAA(stop of α) to 0.75 1.79 (SEQ ID NO:201) GAA (Glu)*The T2/T1 ratio and T2 value are relative numbers, normalized to thewild-type enzyme.

Example 10 Analysis of the Mutations Found in Mutant Sma3002

Sma3002 (SEQ ID NO:140), a first generation mutant identified in EXAMPLE6, displayed significant improvements in both T2/T1 ratio and T2 value.It contained four point mutations, one of which included the fusionmutation explored in detail in EXAMPLE 9. To investigate the effect ofthese mutations individually, two more mutants (α-Y271C and β-Q2R) wereconstructed.

Using methodology previously followed, single point mutations wereintroduced into the wild-type plasmid using uniquely designed primers,as shown below. As in previous EXAMPLES, the nucleotide shown incapitalized, boldface lettering in each primer shows the location of thespecific mutation to be introduced. TABLE 12 Synthesis ofSma3002-Derived Mutants, Each Mutant Containing a Single Point MutationFound in Sma3002 Mutant Mutation Forward Primer Reverse Primer 7A-C1α-Y271C 7A-C1-F1: 7A-C1-R1: 5′-gcg ctg atg 5′-ctt gct ctc ggc tGt tcggag cga aCa gcc cat agc aag-3′ cag cgc-3′ (SEQ ID NO: 30) (SEQ ID NO:31) 7C-A5 β-Q2R 7C-A5-F1: 7C-A5-R1: 5′-ggc ggt att 5′-g aat ttg cct gtgcGa cag ggt tgt ggt tgt aca acc caa att ctg tCg cac agg c-3′ aat aacgcc-3′ (SEQ ID NO: 32) (SEQ ID NO: 33)

Mutagenesis experiments were carried out using Stratagene QuikChangesite-directed mutagenesis kits (Stratagene, La Jolla, Calif.), asdescribed in Example 3. The T2/T1 ratio and T2 value of these mutantswere measured, as described in EXAMPLE 5. TABLE 13 shows the results.The SEQ ID NO: of the DNA sequence of the enzyme is provided in thefirst column of the Table. TABLE 13 T2/T1 Ratio and T2 Value ofSma3002-Derived Mutants. Each Mutant Containing a Single Point MutationRelative to Sma3002 T2/T1 Mutant Mutation ratio* T2* WT — 1 1 (SEQ IDNO: 1) Sma3002 TAT(α-Tyr271) to TGT(Cys); 2.7 1.6 (SEQ ID NO: 140)TAC(α-Tyr502) to CAC(His); TAA(stop of α) to CAA(Gln); CAA(β-Gln2) toCGA(Arg); TTT(β-Phe11) to TTC(Phe) 7A-C1 TAT(α-Tyr271) to TGT(Cys) 1.010.43 (SEQ ID NO: 204) 7C-A5 CAA(β-Gln2) to CGA(Arg) 1.02 0.95 (SEQ IDNO: 208)*The T2/T1 ratio and T2 value are relative numbers, normalized to thewild-type enzyme.Those mutants shown in bold text contain a single point mutation,derived from Sma3002.The α-Y271C mutation decreased the T2 value, but failed to change theT2/T1 ratio. Thus, this mutation decreased the k_(cat) of the enzyme.The other mutation, β-Q2R, did not appear to significantly affect eitherthe T2 or T2/T1 ratio.

To determine whether any of the 3 non-fusion mutations in Sma3002 (SEQID NO:140) acted in concert to increase the enzyme's stability in thepresence of 1,3-propanediol and glycerol, three additional mutants weremade. In each of these mutants, one of the three non-fusion mutations inSma3002 (α-Y271C, α-Y502H, or β-Q2R) was removed, by introducing thewild-type DNA sequence as a single point mutation. This resulted inthree Sma3002-derived mutants, each containing 2 of the originalnon-fusion mutations present in Sma3002. Single point mutations wereintroduced into the Sma3002 plasmid, using the Stratagene QuikChangesite-directed mutagenesis kit, as described in EXAMPLE 3, and theprimers shown in TABLE 14 below. TABLE 14 Synthesis of Sma3002-DerivedMutants, Each Mutant Containing Four Point Mutations Found in Sma3002Mutation Mutant Removed Forward Primer Reverse Primer 8-C9 α-Y271C8-C9-F1 8-C9-R1 (SEQ ID NO: 34) (SEQ ID NO: 35) 9-D7 α-Y502H 9-D7-F19-D7-R1 (SEQ ID NO: 36) (SEQ ID NO: 37) 10-G6 β-Q2R 10-G6-F1 10-G6-R1(SEQ ID NO: 38) (SEQ ID NO: 39)

The T2/T1 ratio and T2 values of these mutants were measured asdescribed in EXAMPLE 5. TABLE 15 shows the results of this analysis. TheSEQ ID NO: of the DNA sequence of the enzyme is provided in the firstcolumn of the Table. TABLE 15 T2/T1 Ratio and T2 Value ofSma3002-Derived Mutants, Each Mutant Containing Four Point MutationsMutation T2/T1 Mutant Removed ratio* T2* WT — 1 1 (SEQ ID NO: 1) Sma3002— 2.7 1.6 (SEQ ID NO: 140) 8-C9 α-Y271C 1.3 1.85 (SEQ ID NO: 212) 9-D7α-Y502H 0.98 1.57 (SEQ ID NO: 215) 10-G6 β-Q2R 2.56 1.55 (SEQ ID NO:218)*The T2/T1 ratio and T2 value are relative numbers, normalized to thewild-type enzyme. Those mutants shown in bold text contain threemutations and were derived from Sma3002.

Example 11 Third Round Mutagenesis by Addition of Some First GenerationMutations to Second Generation Mutants

Although substantial improvement was made in the previous EXAMPLEStoward improving the total turnover of the GDH enzyme, as measured byvalues reported herein as T2, a third round of mutagenesis wasperformed. In these reactions, select point mutations from the firstgeneration mutants were introduced into the two second generationmutants showing the greatest improvement in T2 values (i.e., 1-E1 and8-C9). This was accomplished by first purifying the 1-E1 and 8-C9 mutantplasmids from the host cells. Then, the single amino acid substitutionmutation found in Xba3007, Xba3029, or 4BR1001 was introduced into theseplasmids, using the primers shown below in TABLE 16 and the StratageneQuikChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.),as described in EXAMPLE 3. TABLE 16 Synthesis of Third GenerationMutants 3^(rd) “Parent” Generation Mutant Mutants Forward Primer ReversePrimer 15-E4 8-C9 + 2-F4-F1 2-F4-R1 4BR1001 (SEQ ID NO: 20) (SEQ ID NO:21) 18-D7 8-C9 + 12-B1-F1 12-B1-R1 Xba3007 (SEQ ID NO: 22) (SEQ ID NO:23) 21-D10 1-E1 + 12-B1-F1 12-B1-R1 Xba3007 (SEQ ID NO: 22) (SEQ ID NO:23) 17-F6 8-C9 + 16-H5-F1 16-H5-R1 Xba3029 (SEQ ID NO: 24) (SEQ ID NO:25) 20-B9 1-E1 + 16-H5-F1 16-H5-R1 Xba3029 (SEQ ID NO: 24) (SEQ ID NO:25)

The T2/T1 ratio and T2 value of these third generation mutants weremeasured as described in EXAMPLE 5. TABLE 17 shows the results. The SEQID NO: of the DNA sequence of the enzyme is provided in the first columnof the Table. TABLE 17 T2/T1 Ratio and T2 Value of Third GenerationMutants T2/T1 Mutant Mutation ratio* T2* WT — 1 1 (SEQ ID NO: 1) Sma3002TAT(α-Tyr271) to TGT(Cys); 2.7 1.6 (SEQ ID NO: 140) TAC(α-Tyr502) toCAC(His); TAA(stop of α) to CAA(Gln); CAA(β-Gln2) to CGA(Arg);TTT(β-Phe11) to TTC(Phe) Xba3007 ACC(γ-Thr53) to GCC(Ala) 4.3 0.8 (SEQID NO: 40) Xba3029 CTC(α-Leu509) to TTC(Phe) 3.4 0.9 (SEQ ID NO: 44)4BR1001 GGC(α-Gly216) to GGG(Gly); 1.9 1.1 (SEQ ID NO: 171)GTG(α-Val224) to CTG(Leu) 1-E1 Pure fusion, TAA(stop of α) 0.72 1.82(SEQ ID NO: 198) to CAA(Gln) 8-C9 TAC(α-Tyr502) to CAC(His); 1.30 1.85(SEQ ID NO: 212) TAA(stop of α) to CAA(Gln); CAA(β-Gln2) to CGA(Arg);TTT(β-Phe11) to TTC(Phe) 21-D10 TAA(stop of α) to CAA(Gln); 3.85 1.91(SEQ ID NO: 221) ACC(γ-Thr53) to GCC(Ala) 20-B9 CTC(α-Leu509) toTTC(Phe); 3.86 1.95 (SEQ ID NO: 225) TAA(stop of α) to CAA(Gln) 18-D7TAC(α-Tyr502) to CAC(His); 5.38 1.30 (SEQ ID NO: 229) TAA(stop of α) toCAA(Gln); CAA(β-Gln2) to CGA(Arg); TTT(β-Phe11) to TTC(Phe);ACC(γ-Thr53) to GCC(Ala) 17-F6 TAC(α-Tyr502) to CAC(His); 3.63 1.77 (SEQID NO: 233) CTC(α-Leu509) to TTC(Phe); TAA(stop of α) to CAA(Gln);CAA(β-Gln2) to CGA(Arg); TTT(β-Phe11) to TTC(Phe) 15-E4 TAC(α-Tyr502) toCAC(His); 3.95 1.66 (SEQ ID NO: 237) TAA(stop of α) to CAA(Gln);CAA(β-Gln2) to CGA(Arg); TTT(β-Phe11) to TTC(Phe); GTG(α-Val224) toCTG(Leu)*The T2/T1 ratio and T2 value are relative numbers, normalized to thewild-type.Those mutants shown in bold text are third generation mutants.Overall, there was substantial improvement in enzyme stability in thethird generation mutants. All mutants possessed improved stability(T2/T1 ratio) and total turnover number (T2), relative to the best firstgeneration mutant (i.e., Sma3002 (SEQ ID NO:140)). In fact, mutants20-B9 and 21-D10 had T2 values greater than all mutants previouslygenerated.

Example 12 Biochemical Characterization of Some Second and ThirdGeneration Mutants

To further characterize selected second and third generation mutants,the inactivation rate constants for these mutants in the presence of 10mM glycerol and 50 mM 1,3-propanediol were determined, using the methodsdescribed in the EXAMPLE 4. These results are summarized in TABLE 18, asshown below. TABLE 18 Inactivation Rate Constants of Some Second andThird Generation Mutants T2/T1 Inactivation rate Decrease of Mutantratio* constant (min⁻¹) Inactivation (fold) WT 1 0.85 1 Sma3002 2.7 0.175.1 1-E1 0.72 1.06 0.8 8-C9 1.3 0.41 2.1 15-E4 3.95 0.12 7.1 18-D7 5.380.04 21.3 20-B9 3.86 0.11 7.7 21-D10 3.85 0.09 9.4As expected, the results were consistent with the T2/T1 ratiomeasurements.

Since one application for improved GDHs is for 1,3-propanediolbioproduction, it is important to determine the total turnover number ofthe mutants in the presence of high concentrations of 1,3-propanediol.The assays described above had been performed in the presence of only 50mM. In the late phase of fermentation for 1,3-propanediol bioproduction,the 1,3-propanediol concentration can reach up to 1 M; thus, a secondtwo-point assay was conducted. Specifically, T2 values were alsomeasured in the presence of 10 mM glycerol and 600 mM 1,3-propanediolfor several promising mutants. TABLE 19 summarizes the results: TABLE 19T2 values measured in the presence of 600 mM 1,3-propanediol forSelected Second and Third Generation Mutants Mutant T2_((600 mM)) WT(SEQ ID NO: 1) 1 Sma3002 (SEQ ID NO: 140) 3.3 1-E1 (SEQ ID NO: 198) 2.28-C9 (SEQ ID NO: 212) 3.1 15-E4 (SEQ ID NO: 237) 4.0 18-D7 (SEQ ID NO:229) 4.0 20-B9 (SEQ ID NO: 225) 4.1 21-D10 (SEQ ID NO: 221) 4.3Results were somewhat similar to those obtained for T2_((50mM)),although a few mutants performed better than would have been predicted.The T2_((600mM)) of Sma3002 was 3.3-fold higher than that of thewild-type enzyme. Each of the third generation mutants (15-E4, 18-D7,20-B9, and 21-D10) showed further improvements in T2_((600mM)), relativeto Sma3002. The results suggested that these third generation mutantswith higher T2_((600 mM)) are more resistant to higher concentrations of1,3-propanediol. It is expected that these mutants will be very usefulfor 1,3-propanediol bioproduction.

Example 13 One-Point High Throughput Screening Assay to Measure TotalEnzyme Turnover Number in the Presence of High Concentrations of1,3-Propanediol

When 1,3-propanediol concentration is higher than about 300 mM, theinactivation of GDH occurs almost immediately after the reaction isinitiated. Under these conditions, one cannot use the high throughputscreening assay described in Examples 4 and 5 to screen mutants becauseT1 cannot be accurately measured. The total enzyme turnover number inthe presence of high concentrations of 1,3-propanediol is one of the keyenzyme kinetic parameters that is desirable to improve herein. Thistotal enzyme turnover number can be improved by either decreasing therate of inactivation or increasing the k_(cat). In order to screen formutants having improved total enzyme turnover number in the presence ofhigh concentrations of 1,3-propanediol, a modified high throughputscreening assay was been developed.

Briefly, mutant cells were grown and permeabilized in 96-well plates, asdescribed in EXAMPLE 5. Aliquots (8 μL) of permeabilized cells weretransferred into 96-well reaction plates using a Biomek 2000 robot(Beckman-Coulter, Fullerton, Calif.). Reaction at room temperature wasinitiated by addition to the cells of 40 μL of substrate containing 24μM coenzyme B₁₂, 12 mM glycerol, and 720 mM 1,3-propanediol in 0.1 Mpotassium-HEPES buffer, pH 8, using Qfill2 (Genetix, New Milton,Hampshire, UK). After incubating the plate at room temperature forapproximately 70 min, a 12.5 μL aliquot of the reaction was transferredinto a second plate whose wells contained 12.5 μL of3-methyl-2-benzothiazolinone (MBTH) in 0.4 M glycine-HCl, pH 2.7, usinga Biomek 2000 robot (Beckman-Coulter). The 70 min reaction time allowsaccurate measurement of the total turnover number. The concentration ofthe product, 3-HP, was determined as described in EXAMPLE 5. The totalenzyme turnover number (T(600)) was estimated by measuring theabsorbance at 670 nm using a Spectramax 160 plate reader (MolecularDevices, Sunnyvale, Calif.).

This one-point colorimetric assay is simple to execute, since Qfill2 canadd substrate to one 96-well plate within 15 sec. Further, the assay canprovide higher throughput capability relative to the assay described inEXAMPLES 4 and 5. Comparative results of assays performed in EXAMPLE 5and in the present example are shown in Table 20 for wild-type GDH andfor several mutants having different enzyme kinetic parameters. TABLE 20Comparison of T2/T1 and T(600) Values Sample T1 T2 T2/T1 T(600)Background 0.2 0.2 1 0.82 WT 0.25 1.00 4 0.10 1 0.37 2.08 5.6 0.46 20.19 1.76 9.3 0.41 3 0.09 0.98 11.9 0.11 4 0.51 1.72 3.4 0.23

These results demonstrated that each assay screens for different kineticparameters. Those mutants identified by the screening assay described inthe present Example are more resistant to the higher concentrations of1,3-propanediol and generally have improved stability and reasonablevalues for k_(cat).

Example 14 Correlation Between Enzyme Structure and Mutations

In this Example, the position of mutations having an increased T2 orincreased T2/T1 ratio relative to the wild-type GDH are examined, withrespect to the 3-dimensional crystal structure of GDH. This permittedthe identification of regions within the dehydratase that could beconsidered mutational “hot spots”, where the mutations frequently leadto improved reaction kinetics (such that the rate of inactivation wasreduced). Alternative sequence modifications in these regions wouldlikely result in additional mutants having improvements in reactionkinetics.

Position of Mutations Correlated to 3-Dimensional Structure

The three-dimensional crystal structure of substrate-free formdehydratase has been determined by X-ray crystallography (Liao et al.,J. Inorganic Biochem. 93 (1-2): 84-91 (2003)); additionally, thestructure of the enzyme in complex with 1,2-propanediol has also beenreported (Yamanishi et al., Eur. J. Biochem. 269:4484-4494 (2002)).Based on these structures, mutations that led to an improvement ineither the T2/T1 ratio or T2 (from Examples 6-12 were mapped onto aschematic diagram of each GDH subunit. More specifically, FIG. 3 showsthe distribution of mutants containing a single-point mutation (relativeto wild-type GDH) on the α-subunit (representing 88% of all single-pointmutations). In contrast, FIG. 4 shows the distribution of mutantscontaining multiple-point mutations (relative to wild-type GDH) on theα-subunit; this subunit contained 58% of the total multiple-pointmutations, with the remaining mutations distributed between the β- (31%)and γ- (11%) subunits, respectively.

Hot Spots Identified from the Positive Hits

Although mutations within both the single-point and multiple-pointmutants are located in all three subunits of GDH and are distributedthroughout the large α-subunit, those mutants containing single-pointmutations and those mutants containing multiple-point mutationsdisplayed two common hot spots within the α-subunit.

One mutation hotspot is located on the second α helix (residues 62-70)from the N-terminal of the α-subunit. There are five single-pointmutants found in this region: 3 of them have mutations at residue 62with different amino acid substitutions, 1 has a mutation at residue 65,and 1 has a mutation at residue 70 (FIG. 3). Six mutation sites from themultiple-point mutants are also located on this helix: 3 are at residue62 and 1 each at residues 63, 67 and 70, respectively (FIG. 4).

The second hot spot is the region that includes a portion of the 4^(th)β-strand of the TIM barrel and the following loop and a short helix(residues 224-236) of the α-subunit. This hot spot is in the vicinity ofthe active site. There are five single-site mutations found in thisregion. Residue 224 possessed different amino acid substitutions in twomutants. Residue 226 is the mutation site of two identical mutants. Onemutant is on residue 233. Additionally, three mutation sites of themultiple-site mutants are located in this region (residues 226, 231, and236).

Example 15 Site-Saturation Mutagenesis of GDH Mutants

Three mutation sites present in the mutants characterized in Example 9were subjected to site-saturation mutagenesis. Specifically, these siteswere: γ-Thr53 (found in mutant Xba3009), α-Leu509 (found in mutantXba3029) and α-Val224 (found in mutant 4BR1001). To prepare thesaturation mutagenesis libraries, the 1-E1 and 8-C9 mutants (Examples 9and 10, respectively) were purified from their host cells and used astemplates, along with the degenerate primers shown below in Table 21.TABLE 21 Saturation Mutagenesis libraries Saturation Library MutagenesisDegenerate Name Site(s) Template Primer GDH- γ-T53 1-E1 T53-SM-for: SM15′-tg cgg atc tcc cgc cag NNN ctt gag tac cag g-3′ (SEQ ID NO:340) GDH-α-L509 1-E1 L509-SM-for: SM2 5′-ctg cag acc tcg gcc att NNN gat cgg cagttc gag gtg-3′ (SEQ ID NQ:341) GDH- α-V224 8-C9 V224-SM-for: SM3 5′-agctac gcc gag acg NNN tcg gtc tac ggc acc-3′ (SEQ ID NO:342) GDH- α-L509and 1-E1 L509-SM-for: SM4 T53 (SEQ ID NO:341); and T53-SM-for: (SEQ IDNO:340)

Mutagenesis experiments were carried out using the Stratagene QuikChangeMulti Site Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.)according to the manufacturers' instructions, to prepare the GDH-SM1,GDH-SM2, GDH-SM3, and GDH-SM4 libraries. Following electroporation ofthe plasmids into E. coli strain 5K(DE3) (as described in Example 1), 88mutant colonies from each library were screened using the one-point GDHassay to estimate total enzyme turnover number in the presence of highconcentrations of 1,3-propanediol. The best hit from each screen wasthen subjected to DNA sequence analysis. The following Table shows theresults. The SEQ ID NO: of the DNA sequence of the enzyme is provided inthe first column of the Table. TABLE 22 T(600) Values and Mutations inFour Saturation Mutants Mutant Origin of Mutant Mutation T(600)* WT — —1 (SEQ ID NO:1) Sma3002 Example 6 TAT(α-Tyr271) to TGT(Cys); 3.3 (SEQ IDNO:140) TAC(α-Tyr502) to CAC(His); TAA(stop of α) to CAA(Gln);CAA(β-Gln2) to CGA(Arg); TTT(βPhe11) to TTC(Phe) Xba3007 Example 6ACC(γ-Thr53) to GCC(Ala) 0.7 (SEQ ID NO:40) Xba3029 Example 6CTC(αLeu509) to TTC(Phe) 1.4 (SEQ ID NO:44) 4BR1001 Example 6GGC(α-Gly216) to GGG(Gly); 2.4 (SEQ ID NO:171) GTG(α-Val224) to TTG(Leu)8-C9 Example 10 TAC(α-Tyr502) to CAC(His); 3.1 (SEQ ID NO:212) TAA(stopof α) to CAA(Gln); CAA(β-Gln2) to CGA(Arg); TTT(β-Phe11) to TTC(Phe)1-E1 Example 9 TAA(stop of α) to CAA(Gln) 2.2 (SEQ ID NO:198)GDH-SM1-G11 Present TAA(stop of α) to CAA(Gln); 4.3 (SEQ ID NO:301)Example ACC(γ-Thr53) to TCC(Ser) GDH-SM2-B11 Present TAA(st p of α) tCAA(Gln); 4.1 (SEQ ID NO:304) Example CTC(α-Leu509) t TTT(Phe)GDH-SM3-D2 Present GTG(α-Val224) to TTG(Leu); 4.0 (SEQ ID NO:307)Example TAC(α-Tyr502) to CAC(His); TAA(stop of α) to CAA(Gln);CAA(β-Gln2) to CGA(Arg); TTT(β-Phe11) to TTC(Phe) GDH-SM4-H2 PresentTAA(stop of α) to CAA(Gln); 4.1 (SEQ ID NO:310) Example ACC(γ-Thr53) toTGT(Cys)*The T(600) values are relative numbers, normalized to the wild-type.Those mutants shown in bold text are saturation mutants.

All four saturation mutants (GDH-SM1-G11, GSH-SM2-B11, GDH-SM3-D2, andGDH-SM4-H2 [shown in bold text]) showed further improvements in T(600),as compared to the parent mutant genes from which they were derived.Interestingly, in mutant GDH-SM4-H2 a three-base change was identified(i.e., ACC(γ-Thr53) to TGT(Cys)). This type of mutation is extremelydifficult to generate using error-prone PCR.

Example 16 The Recombinogenic Extension Method Using Unpaired Primers toGenerate GDH Mutants

Despite significant improvements in the GDH rate of inactivation in thepresence of glycerol and 1,3-propanediol using random mutagenesis,rational design mutagenesis, and saturation mutagenesis (Examples 6,8-11, and 15), further improvements were desirable for industrialapplications. Thus, 24 glycerol dehydratase mutants from Examples 6, 9,10, and 15 were utilized as parent templates in a single recombinogenicextension reaction using the unpaired primers method (U.S. 60/360,279).

Attaching a Short Flanking DNA Fragment to the 5′ or 3′ End of ParentGenes

A short flanking DNA fragment was attached to the 5′ or 3′ end of theparent genes by PCR, which was subsequently used as the binding sitesfor the recombinogenic extension method using unpaired primers. Plasmidscontaining GDHs were purified from host cells and used as templates.

Specifically, forward primer GDHM-F1 (SEQ ID NO: 343) and reverse primerGDHM-R1 (SEQ ID NO: 344) were used to amplify the following templategenes in standard high fidelity PCR reactions using the Expand HighFidelity PCR System (Roche Applied Science, Indianapolis, Ind.): 1-E1(SEQ ID NO:198), wild type GDH (SEQ ID NO:1), Xba3023 (SEQ ID NO:182),Xba3010 (SEQ ID NO:175), 8-C9 (SEQ ID NO:212), 4BR1001 (SEQ ID NO:171),Xba3015 (SEQ ID NO:147), Xba3008 (SEQ ID NO:151), Sma3003 (SEQ IDNO:143), Xba3016 (SEQ ID NO:155) and Xba3020 (SEQ ID NO:159). Theresulting PCR products were then mixed together in an equal molar ratio,and designated as “mixture-1”.

Forward primer GDHM-F2 (SEQ ID NO:345) and reverse primer GDHM-R2 (SEQID NO:346) were used to amplify the following genes in a similar manner:Xba3007 (SEQ ID NO:40), Xba3029 (SEQ ID NO:44), Xba3025 (SEQ ID NO:52),Sma3009 (SEQ ID NO:179), Sma3010 (SEQ ID NO:175), Sma3008 (SEQ IDNO:151), RsrII001 (SEQ ID NO:136), PpuMI002 (SEQ ID NO: 128), KG005 (SEQID NO:253), GDH-SM1-G11 (SEQ ID NO:301), GDH-SM2-B11 (SEQ ID NO:304),GDH-SM3-D2 (SEQ ID NO:307) and GDH-SM4-H2 (SEQ ID NO:310). The resultingPCR products were then mixed together in an equal molar ratio, anddesignated as “mixture-2”.

The amplified products were purified from agarose gels using a QiagenDNA extraction kit (Qiagen, Valencia, Calif.), and then used as theparent templates for the recombinogenic extension method with unpairedprimers.

Making Recombinogenic Mutant Products Using the Unpaired Primers Method

Recombinogenic products (i.e., GDH mutant genes) were made from theabove-mentioned 24 parent templates using two primers: PADH316F1 (SEQ IDNO:29) and T7T (SEQ ID NO:30). PADH316F1 anneals with the 5′ end ofthose templates in “mixture-1” (i.e., 1-E1, wild type GDH, Xba3023,Xba3010, 8-C9, 4BR1001, Xba3015, Xba3008, Sma3003, Xba3016 and Xba3020),due to the addition of the short 5′ DNA fragment produced by SEQ ID NO:343. However, primer PADH316F1 does not bind to the 5′ end of the“mixture-2” templates. In like manner, primer T7T anneals to the 3′ endof the “mixture-2” templates (i.e., Xba3007, Xba3029, Xba3025, Sma3009,Sma3010, Sma3008, RsrII001, PpuMI002, KG005, GDH-SM1-G11, GDH-SM2-B11,GDH-SM3-D2 and GDH-SM4-H2), but does not bind to the 3′ end of the“mixture-1” templates.

The following reaction mixture was assembled for the reaction: 10 ng“mixture-1”, 10 ng “mixture-2”, 200 μM each dNTP, 1×PCR Buffer (with 1.5mM MgCl₂, as the final concentration), 286 nM pADH316F1, 286 nM T7T,0.625 U HotStarTaq (Qiagen, Valencia, Calif.), and dH₂O to 25 μl.Thermal cycling conditions were: 95° C. denaturation for 2 min; followedby 60 cycles of 30 sec at 95° C., 1 sec+1 sec per cycle at a gradientbetween 63-69° C.; 72° C. final extension for 7 min; and hold at 4° C.An Eppendorf Mastercycler gradient5331 (Eppendorf Scientific, Inc.,Westbury, N.Y.) was used for the thermal cycling reactions.

Since the two primers used in the reaction do not match the 5′ and 3′ends of any template simultaneously, none of the parent templates can beamplified during the reaction. However, any recombinant DNA productpossessing both the 5′ and 3′ ends would be amplified during subsequentthermal cycles. Following the unpaired primer reaction, the reactionmixtures were loaded onto an agarose gel. At about 66-67° C., a largeamount of 2.7 kB PCR products were obtained from the unpaired primerreaction. Products of this size were expected since the original parentmolecules used as templates were themselves about 2.7 kB.

Making and Screening the Recombinogenic GDH Mutant Library

The recombinant GDH DNA products (approximately 2.7 kB) were purifiedfrom the gel using a Qiagen DNA extraction kit, digested with Xba I andHind III, and then ligated into the XbaI-Hind/III-digested pGD20 vector(Example 1). The mutant library was obtained by transformation of theligation mixture into E. coli strain 5K(DE3) by electroporation, asdescribed in Example 1. The library size was over 0.3 million coloniesper ligation reaction.

Between 6,000-7,000 mutant colonies were picked from agarose plates andscreened using the one-point high throughput screening assay, asdescribed in Example 13. Following primary screening, putative hits wereconfirmed by a follow-up confirmation assay. Briefly, each putative hitwas re-assayed in 8 wells. Results from each individual clone wereanalyzed statistically to obtain the mean and standard deviation forT(600). These results were compared to the wild-type GDH enzyme.

Additionally, several hits were further investigated using the two-pointassay (Example 5) to roughly estimate the change of initial reactionrate and inactivation for these hits.

Table 23 summarizes the results of these various assays for severalrecombinogenic GDH mutants. TABLE 23 Characterization of RecombinogenicGDH Mutants Obtained in the Recombinogenic Extension Method usingUnpaired Priers T2/T1 Mutant T(600)* T1* T2* ratio* WT 1.0 1.00 1.001.00 SHGDH37 6.6 0.51 2.79 5.47 SHGDH51 6.2 0.19 2.63 13.84  SHGDH12 5.90.64 3.46 5.41 SHGDH22 5.8 0.50 3.42 6.84 SHGDH38 5.7 — — — SHGDH24 5.61.05 2.29 2.18 SHGDH43 5.6 0.22 2.11 9.59 SHGDH25 5.1 — — — SHGDH29 4.60.49 2.14 4.37*The T(600), T1, T2, and the T2/T1 ratio values are relative numbers,normalized to the wild-type. Those mutants shown in bold text werecreated using the recombinogenic extension method using unpairedprimers.

Although different recombinant GDH mutants displayed different enzymekinetics (despite having similar T(600) values), this merely reflectsdifferences in the parameters that each assay measures. For example,mutants SHGDH24 and SHGDH43 possessed identical T(600) values, butSHGDH24 had only a slightly improved T1 value compared with wild typewhile SHGDH43 had a largely reduced T1. In the case of SHGDH24, thek_(cat) of the enzyme was not decreased; in contrast, SHGDH43 had ak_(cat) that was greatly reduced. In either case, the GDH enzymestability of both SHGDH24 and SHGDH43 was increased, thus enablingimprovement in the total enzyme turnover number.

SHGDH51 showed the greatest improvement for stability among thesemutants, but this mutant did not have the highest total enzyme turnovernumber because its k_(cat) was largely decreased. SHGDH37 displayed thegreatest improvement for T(600) among these mutants, but its T2 value isless than that of SHGDH12, indicating that SHGDH37 is more resistant tohigh concentrations of 1,3-propanediol than SHGDH12.

In any case, however, the results clearly demonstrated substantialimprovement in GDH stability, due to the creation of new mutants usingthe unpaired primer method. Specifically, the best mutant obtained viarandom mutagenesis had a T(600) of 3.3 (i.e., mutant Sma3002; SEQ IDNO:140); rational combination of mutants identified through randommutagenesis and screening or semi-random combination of these mutationsusing site-saturation mutagenesis yielded a mutant with a maximum T(600)of 4.3 (i.e., mutant GDH-SM1-G11; SEQ ID NO:301). Thus, it is concludedthat the random recombination approach using the recombinogenicextension method using unpaired primers is a more powerful techniquethan previous rational and semi-random approaches.

Sequence Analysis of the Mutant Genes

Plasmid DNA was purified from the recombinant GDH mutants listed inTable 23 and the entire GDH gene in each was sequenced. Analysis of themutants, followed by comparison to the original wild type GDH genesequence, indicated that these recombinant mutant genes contained thesingle base substitution mutations (point mutations) that appear inTable 24. The SEQ ID NO: of the DNA sequence of the enzyme is providedin the first column of the Table: TABLE 24 DNA sequence analysis ofRecombinogenic GDH Mutants Strain Mutations SHGDH12 GTT(α-Val74) toATT(Ile); (SEQ ID NO:319) GTG(α-Val224) to TTG(Leu); CGC(α-Arg425) toCGT(Arg); TAC(α-Tyr502) to CAC(His); TAA(stop of α) to CAA(Gln);CAA(β-Gln2) to CGA(Arg); TTT(β-Phe11) to TTC(Phe); AAA(β-Lys14) toAGA(Arg) SHGDH22 GGC(α-Gly216) to GGG(Gly); (SEQ ID NO:322)GTG(α-Val224) to TTG(Leu); CAG(α-Gln337) to CAA(Gln); CGC(α-Arg533) toGGC(Gly); ACC(α-Thr553) to ACG(Thr); TAA(stop of α) to CAA(Gln);ATC(γ-Ile21) to ACC(Thr); CTG(γ-Leu137) to CTA(Leu) SHGDH24CGT(α-Arg134) to CGC(Arg); (SEQ ID NO:328) GGC(α-Gly216) to GGG(Gly);GTG(α-Val224) to TTG(Leu); AGC(α-Ser481) to AGT(Ser); ACC(α-Thr553) toACG(Thr); TAA(stop of α) to CAA(Gln) SHGDH25 ATG(α-Met62) to CTG(Leu);(SEQ ID NO:334) GTG(α-Val124) to GCG(Ala); GGC(α-Gly216) to GGG(Gly);GTG(α-Val224) to TTG(Leu); TAA(stop of α) to CAA(Gln) SHGDH29GCC(α-Ala376) to GCT(Ala); (SEQ ID NO:337) CTC(α-Leu509) to TTT(Phe);ACC(α-Thr553) to ACG(Thr); TAA(stop of α) to CAA(Gln); CAG(γ-Gln101) toCGG(Arg) SHGDH37 GTG(α-Val224) to TTG(Leu); (SEQ ID NO:313)TAC(α-Tyr502) to CAC(His); TAA(stop of α) to CAA(Gln); CAA(β-Gln2) toCGA(Arg); TTT(β-Phe11) to TTC(Phe); GAG(γ-Glu35) to AAG(Lys) SHGDH38GTG(α-Val224) to TTG(Leu); (SEQ ID NO:325) TAC(α-Tyr502) to CAC(His);TAA(stop of α) to CAA(Gln); CAA(β-Gln2) to CGA(Arg); TTT(β-Phe11) toTTC(Phe); GGG(β-Gly19) to GAG(Glu); GAA(β-Glu64) to GAG(Glu);CTT(β-Leu67) to CTC(Leu); AAT(γ-Asn72) to AGT(Ser) SHGDH43 GGC(α-Gly216)to GGG(Gly); (SEQ ID NO:331) GTG(α-Val224) to TTG(Leu); GAG(α-Glu240) toGAA(Glu); GTG(α-Val301) to GTA(Val); ACC(α-Thr553) to ACG(Thr); TAA(stopof α) to CAA(Gln); AAA(β-Lys166) to AGA(Arg); AAA(β-Lys173) to GAA(Glu);AGC(γ-Thr53) to TCC(Ser) SHGDH51 TTC(α-Phe339) to GTC(Val); (SEQ IDNO:316) CGC(α-Arg346) to CGG(Arg); ACC(α-Thr553) to ACG(Thr); TAA(stopof α) to CAA(Gln); CCC(β-Pro84) to CCT(Pro); ACC(γ-Thr53) to GCC(Ala)

Interestingly, all of the mutants (SEQ ID NOs:313, 316, 319, 322, 325,328, 331, 334, 337) contained an α-β fusion mutation (as originallydiscovered in the Sma3002 and Xba3009 mutants [Example 6]), indicatingthat this mutation is very important for T(600) value improvement.SHGDH43 contained mutations from four different parent genes (i.e.,4BR1001, 1-E1, Xba3008 and GDH-SM1-G11), in addition to four newlycreated mutations. This indicated that at least four crossovers occurredamong the four different parent genes during the recombinogenic PCR.SHGDH25 and SHGDH51 contained mutations from three parent genes (i.e.,Sma3003, 4BR1001 and 1-E1 for SHGDH25; RsrII001, 1-E1 and Xba3007 forSHGDH51), in addition to several newly created mutations.

1. A nucleic acid sequence encoding a B₁₂-dependent mutant dehydrataseselected from the group consisting of SEQ ID NOs: 40, 44, 48, 52, 56,60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120,124, 128, 132, 136, 140, 143, 147, 151, 155, 159, 163, 167, 171, 175,179, 182, 186, 189, 192, 195, 198, 201, 204, 208, 212, 215, 218, 221,225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277,281, 285, 289, 293, 297, 301, 304, 307, 310, 313, 316, 319, 322, 325,328, 331, 334, and
 337. 2. A B₁₂-dependent mutant dehydratase encoded bythe nucleic acid sequence selected from the group consisting of: SEQ IDNOs: 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100,104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 143, 147, 151, 155,159, 163, 167, 171, 175, 179, 182, 186, 189, 192, 195, 198, 201, 204,208, 212, 215, 218, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257,261, 265, 269, 273, 277; 281, 285, 289, 293, 297, 301, 304, 307, 310,313, 316, 319, 322, 325, 328, 331, 334, and
 337. 3. A nucleic acidsequence encoding a B₁₂-dependent mutant dehydratase comprising an α-βsubunit fusion selected from the group consisting of SEQ ID NOs: 140,179, 186, 189, 192, 195, 198, 201, 212, 215, 218, 301, 304, 307, 310,313, 316, 319, 322, 325, 328, 331, 334, and
 337. 4. A nucleic acidsequence encoding a B₁₂-dependent mutant dehydratase comprising an α-βsubunit fusion selected from the group consisting of SEQ ID NOs: 313,322, and
 328. 5. The nucleic acid sequence of claim 3, furthercomprising a linker sequence between the α and β subunits of the α-βsubunit fusion, wherein the linker sequence is selected from the groupconsisting of SEQ ID NO: 18 and SEQ ID NO:
 19. 6. A method for creatingB₁₂-dependent dehydratase mutants having improved reaction kinetics,comprising: (a) contacting a B₁₂-dependent dehydratase holoenzymecomprising hotspot 1 or hotspot 2 with a mutating agent; (b) screeningthe mutants produced in step (a) for improved k_(cat) and/or stability;and (c) repeating steps (a) and (b).